Programmable genotoxic agents and uses therefor

ABSTRACT

The compositions and methods disclosed herein provide heterobifunctional programmable genotoxic compounds that can be designed to kill selected cells present in a heterogenous cell population. The present compounds comprise a first agent that inflicts damage on cellular DNA, and a second agent that attracts a macromolecular cell component such as a protein, which in turn shields genomic lesions from repair. Unrepaired lesions therefore persist in the cellular genome and contribute to the death of selected cells. In contrast, lesions formed in nonselected cells, which lack the cell component, are unshielded and thus are repaired. As a result, compounds described herein are less toxic to nonselected cells. Compounds of this invention can be designed to cause the selective killing of transformed cells, viral-infected cells and the like.

GOVERNMENT SUPPORT

Work described herein was supported by Federal Grant No. 5R35-CA52127,awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to compounds and methods for theselective destruction of cells in a heterogenous cell population. Thecompounds feature, in pertinent part, a genotoxic agent that damagescellular DNA.

BACKGROUND OF THE INVENTION

Frequently, a need arises in biological investigations and clinical orveterinary practice for selectively killing a subpopulation of cells ina heterogenous cell population. For example, to attain a strain orculture of cells having desirable characteristics, available in vitrotechniques can be applied for selectively killing a subpopulation ofcells in a heterogenous cell population that comprises cells thatpossess a desired characteristic. In this manner, cells that haveundesirable characteristics can be eliminated from the population.Hybridoma cell lines producing desired monoclonal antibodies and stablegenetic transfectant cell lines expressing the products of heterologouscloned genes are customarily established in this manner. See generally,Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. 1989). Amixed population of cells comprising the desired hybridoma ortransfectant is maintained in culture for a period of time in thepresence of one or more genotoxic drugs, such as aminopterin ormethotrexate. The desired cells are resistant to the genotoxic effectsof the drug employed. In contrast, other cells in the population aresusceptible to the drug and fail to survive. These techniques rest onthe creation of cells having a defined phenotype that confers resistanceto a particular, preselected genotoxic drug. Thus, although significantadvances in biology and biotechnology have been achieved through the useof these techniques, limits remain to their flexibility.

Another general context in which practitioners desire to kill cellsselectively involves heterogenous cell populations comprising cells oftwo or more phylogenetically different species of organisms. Here, itmay be desirable to destroy selectively the cells of one species whilepreserving viability of another. In this manner, a desired species canbe enriched in the population or an offensive species, such as aninfectious agent, can be removed. Here again, the desired objective isoften accomplished by treating the cell population with a drug, such asan antibiotic, antiviral, antifungal or antiparasitic drug, to which theundesired species is susceptible. Cells of the undesired species succumbto the effects of the drug and die. Conversely, the desired species(e.g., a human or other host animal) must have the capacity to resistthe chosen drug. Although a wide choice of drugs useful for suchpurposes has historically been available, recent reports have documentedthe appearance of drug resistance in undesirable species. For example,resistant strains of the organisms responsible for septic woundinfections, hospital-acquired infections, tuberculosis, malaria,dysentery and a host of other contagious diseases have arisen in recentyears. Harrison's Principles of Internal Medicine, Part 5 InfectiousDiseases, Ch. 78, 79, and 83-88 (12th ed. 1991). The emergence of suchstrains greatly complicates the treatment of infection, and limitschoices available to the practitioner.

The need to manage or alleviate cancer provides yet another generalsetting in which practitioners require means for selectively killingcells in a heterogenous cell population. Here, the population comprisesnormal and neoplastic (malignant or transformed) cells in anindividual's tissues. Cancer arises when a normal cell undergoesneoplastic transformation and becomes a malignant cell. Transformed(malignant) cells escape normal physiologic controls specifying cellphenotype and restraining cell proliferation. Transformed cells in anindividual's body thus proliferate, forming a tumor (also referred to asa neoplasm). When a neoplasm is found, the clinical objective is todestroy malignant cells selectively while mitigating any harm caused tonormal cells in the individual undergoing treatment. Currently, threemajor genotoxic approaches are followed for the clinical management ofcancer in humans and other animals. Surgical resection of solid tumors,malignant nodules and or entire organs may be appropriate for certaintypes of neoplasia. For other types, e.g., those manifested as soluble(ascites) tumors, hematopoeitic malignancies such as leukemia, or wheremetastasis of a primary tumor to another site in the body is suspected,radiation or chemotherapy may be appropriate. Either of these techniquesis also commonly used as an adjunct to surgery. Harrison's Principles ofInternal Medicine, Part 11 Hematology and Oncology, Ch. 296, 297 and300-308 (12th ed. 1989).

Chemotherapy is based on the use of drugs that are selectively toxic tocancer cells. Id. at Ch. 301. Several general classes ofchemotherapeutic drugs have been developed, including drugs thatinterfere with nucleic acid synthesis, protein synthesis, and othervital metabolic processes. These are generally referred to asantimetabolite drugs. Treatment regimes typically attempt to ensureinactivation of a particular pathway in cancer cell metabolism bycoadministering two or more suitable antimetabolite drugs. Other classesof chemotherapeutic drugs inflict damage on cellular DNA. Drugs of theseclasses are generally referred to as genotoxic. The repair of damage tocellular DNA is an important biological process carried out by a cell'senzymatic DNA repair machinery. Unrepaired lesions in a cell's genomecan impede DNA replication or impair the replication fidelity of newlysynthesized DNA. Thus, genotoxic drugs are generally considered moretoxic to actively dividing cells that engage in DNA synthesis than toquiescent, nondividing cells. In many body tissues, normal cells arequiescent and divide infrequently. Thus, greater time between rounds ofcell division is afforded for the repair of damage to cellular DNA innormal cells. In this manner, practitioners can achieve some selectivityfor the killing of cancer cells. Many treatment regimes reflect attemptsto improve selectivity for cancer cells by coadministeringchemotherapeutic drugs belonging to two or more of these generalclasses.

In some tissues, however, normal cells divide continuously. Thus, skin,hair follicles, buccal mucosa and other tissues of the gut lining, spermand blood-forming tissues of the bone marrow remain vulnerable to theaction of genotoxic drugs. These and other classes of chemotherapeuticdrugs can also cause severe adverse side effects in drug-sensitiveorgans, such as the liver and kidneys. These and other adverse sideeffects seriously constrain the dosage levels and lengths of treatmentregimens that can be prescribed for individuals in need of cancerchemotherapy. Id. at Ch. 301. See also Loehrer and Einhorn (1984), 100Ann. Int. Med. 704-714 and Jones et al. (1985), 52 Lab. Invest. 363-374.Such constraints can prejudice the effectiveness of clinical treatment.For example, the drug or drug combination administered must contact andaffect cancer cells at times appropriate to impair cell survival.Genotoxic drugs are most effective for killing cancer cells that areactively dividing when chemotherapeutic treatment is applied.Conversely, such drugs are relatively ineffective for the treatment ofslow growing neoplasms. Carcinoma cells of the breast, lung andcolorectal tissues, for example, typically double as slowly as onceevery 100 days. Id. at Table 301-1. Such slowly growing neoplasmspresent difficult chemotherapeutic targets.

Moreover, as with the emergence of resistant strains of pathogenicorganisms, transformed cells can undergo further phenotypic changes thatincrease their resistance to chemotherapeutic drugs. Cancer cells canacquire resistance to genotoxic drugs through diminished uptake or otherchanges in drug metabolism, such as those that occur upon drug-inducedgene amplification or expression of a cellular gene for multiple drugresistance (MDR). Id. at Ch. 301. Resistance to genotoxic drugs can alsobe acquired by activation or enhanced expression of enzymes in thecancer cell's enzymatic DNA repair machinery. Therapies that employcombinations of drugs, or drugs and radiation, attempt to overcome theselimitations. The pharmacokinetic profile of each chemotherapeutic drugin such a combinatorial regime, however, will differ. In particular,permeability of neoplastic tissue for each drug will be different. Thus,it can be difficult to achieve genotoxically effective concentrations ofmultiple chemotherapeutic drugs in target tissues.

Needs remain for drugs that can selectively destroy cells in aheterogenous cell population. Particular needs remain for drugs,including genotoxic drugs, that can selectively destroy cells of apathogenic or undesired organism while preserving relatively unimpairedthe viability of cells of a host or desired organism. Still morepoignant needs remain for chemotherapeutic drugs, including genotoxicdrugs, that can selectively destroy neoplastic or virally infected cellsyet not significantly impair the viability of normal healthy cells inthe body of an individual afflicted with cancer or a viral disease.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a heterobifunctionalcompound that is genotoxic to selected cells in a heterogenous cellpopulation. It is an object of this invention to provide aheterobifunctional compound that inflicts genomic lesions on selectedcells in a heterogenous cell population. It is an object of thisinvention to provide a heterobifunctional compound that inflicts genomiclesions and impairs cellular repair of said lesions in selected cells ina heterogenous cell population. It is an object of this invention toprovide a genotoxic agent or drug that can be "programmed" to destroyselected cells that are phenotypically distinguishable from nonselectedcells in a heterogenous cell population. Another object of thisinvention is to expand the range of chemotherapeutic drugs available forthe treatment of infectious and neoplastic diseases. Yet another objectof this invention is to expand the range of infectious and neoplasticdiseases that are susceptible to chemotherapy. These and other objects,along with advantages and features of the invention disclosed herein,will be apparent from the description, drawings and claims that follow.

In one aspect, the invention features a cell membrane permeantheterobifunctional compound suitable for destroying selected cells in aheterogenous cell population. The selected cells possess a cellcomponent, such as a protein, that is absent or is present atsignificantly diminished levels in other, nonselected cells of theheterogenous cell population. Preferably, the cell component isintracellular. Most preferably, it is located within the cell nucleus oris naturally translocated to the nucleus from another intracellularsite. In preferred embodiments, the cell component is a diffusiblemacromolecule having a molecular weight of at least about 25 kDa, morepreferably at least about 40 kDa and still more preferably at leastabout 80 kDa. The present heterobifunctional compound is actively orpassively transported across cell membranes, or diffuses through cellmembranes. Thus, it can internalize within cells. It comprises a firstagent that binds to cellular DNA to form a genomic lesion. The genomiclesion can be formed at a random or site-specific locus in cellular DNA.In certain embodiments, the first agent damages cellular DNA by formingone or more covalent bonds with nucleotide bases, the sugar-phosphateDNA backbone, or both. In other embodiments, the genomic lesion isformed by intercalation of the first agent into cellular DNA.Optionally, the first agent is a precursor that is converted into aDNA-reactive intermediate spontaneously or by exposure to physiologicalconditions, a cellular or secreted enzyme, product or byproduct ofcellular metabolism, ionizing or nonionizing radiation, light energy, orthe like. The genomic lesion so formed by interaction of the first agentwith cellular DNA is potentially repairable by the cell's enzymatic DNArepair machinery.

The first agent is linked to a second agent that binds to the cellcomponent that is preferentially present in selected cells of thepopulation. In some embodiments, the first and second agents are linkedby a covalent bond. In other embodiments, the first and second agentsare linked indirectly by covalent bonds to an organic linker. In stillother embodiments, the first and second agents are linked by noncovalentinteractions, such as electrostatic or hydrophobic interactions. Thus,in certain embodiments, the first and second agents become linked uponor following binding of the first agent to cellular DNA. The secondagent forms a stable complex with the cell component. That is, thesecond agent interacts specifically with the cell component. Interactioncan be noncovalent or covalent, and is energetically favored underintracellular, e.g., nuclear, conditions. As noted, the cell componentis preferably a diffusible macromolecule, such as a protein.Alternatively, it can be a metabolite, ligand or cofactor that isspecifically bound by a protein or another diffusible macromoleculepresent in the cell. In either circumstance, the complex comprises amacromolecular cell component found preferentially in the selectedcells. The second agent thus localizes a sterically large cell componentin the immediate vicinity of the genomic lesion. Preferably, the cellcomponent is large enough to sterically obscure a segment adjacentnucleosides extending from the lesion site for at least about five basepairs, more preferably at least about eight base pairs, still morepreferably at least about twelve base pairs in both the 5' and 3'directions. As a result, the complex between the cell component and thesecond agent is effective for shielding or inhibiting repair of thegenomic lesion formed by the binding of the first agent to cellular DNA.Formation of a sterically large complex at the lesion site hindersaccess by the cell's enzymatic DNA repair machinery. As a result,shielded lesions persist in the genome and prejudice DNA replication,the expression of genes relevant to cell survival, and the like. Thus,the heterobifunctional compounds of the present invention are fatal toselected cells of the heterogenous cell population.

In certain embodiments, the second agent interacts specifically with acell component that is relevant to the survival or proliferation of theselected cells. For example, the second agent can interact with aregulatory protein or enzyme involved in the control of cellproliferation. These include, but are not limited to, oncogene products(e.g., myc, ras, abl, and the like), tumor suppressor gene products(e.g., the nuclear phosphoprotein p53), and proteins that regulateinitiation and progress through the cell cycle (e.g., cyclins andcyclin-dependent kinases). Alternatively, the second agent can interactwith a transcription factor that controls or modulates the expression ofone or more genes that are relevant to metabolic or secretory processescarried out by the selected cell. One such transcription factor isupstream binding factor (UBF), which controls the expression ofribosomal RNA genes and thus is pivotal to the function of the cell'sprotein synthesis machinery. Second agents that specifically interactwith transcription factors preferably mimic or resemble the naturalgenomic binding site for the particular transcription factor. That is,the transcription factor binds to the second agent with an affinity near(e.g., within about 100-fold) or preferably exceeding its affinity forthe natural genomic binding site. Such second agents are referred toherein as "transcription factor decoys". Certain transcription factors,in addition to binding an endogenous genomic binding site, also bind tosoluble ligands. Binding of these transcription factors to their cognateligands modulates binding of the transcription factors to theirendogenous genomic binding sites. That is, ligand binding confers orabrogates ability of the transcription factor to bind its cognategenomic site, or enhances or suppresses its ability to do so. Suchtranscription factors are accordingly referred to herein asligand-responsive transcription factors. They have sometimes beenreferred to in the art as intracellular or nuclear receptors for solubleligands. Second agents that recognize and bind to these transcriptionfactors can mimic an activating or repressing ligand, such as estrogenor an estrogen analog or derivative. Heterobifunctional compoundscomprising transcription factor decoys or ligand mimics thus are doublyfatal to the selected cell.

In another aspect, the present invention provides a method for thedestruction of selected cells in a heterogenous cell population. Theheterogenous cell population can comprise phenotypically distinguishablecells of a single phylogenetic species, or cells of two or moredifferent phylogenetic species. The phylogenetic species can beunicellular or multicellular. The population can comprise cells inculture, cells withdrawn from a multicellular organism (e.g., a bloodsample or tissue biopsy), or cells present in tissue or organs of amulticellular organism. It should be understood that the term"multicellular organism" embraces mammals, including humans. Theheterogenous cell population can comprise cells of both normal andtransformed phenotypes. Thus, the population can comprise neoplastic ormalignant cells. In the present method, selected cells of theheterogenous population are killed. "Selected cells" are phenotypicallydistinguishable from other, nonselected cells in the heterogenouspopulation in that they possess a cell component that is absent or ispresent at significantly diminished levels in nonselected cells. Forexample, the cell component is made or accumulates in the selected cellsto levels that are about 5-fold in excess of the levels of the same or asimilar cell component in nonselected cells. Preferably, the selectedcells possess about a 10-fold excess of the cell component. Morepreferably, the selected cells possess about a 100-fold or higher excessof the cell component. In certain embodiments, the cell component is theexpression product of a cellular or viral oncogene. In certain otherembodiments, the cell component is the expression product of a mutanttumor suppressor gene. In still other embodiments, the cell component isa regulatory or enzymatic element of a nuclear protein complex thatcontrols initiation of or progress through the cell cycle.

The present method involves contacting the heterogenous cell populationwith the cell membrane permeant heterobifunctional compound describedherein. The population is incubated with the compound for a period oftime sufficient for the compound to cross cell membranes and internalizewithin cells, including the selected cells. The first agent of thecompound binds to cellular DNA, inflicting a genomic lesion. As notedabove, the genomic lesion is potentially repairable. In selected cells,the second agent of the compound binds to the cell component, forming acomplex at the genomic lesion site that sterically hinders access to thelesion by the cell's DNA repair machinery thereby inhibiting repair or"shielding" the lesion. As a result, genomic lesions persist in theselected cells and contribute to their demise. That is, the presentcompound is preferentially genotoxic to the selected cells. In contrast,lesions in nonselected cells do not form complexes at the site of thegenomic lesion, or form complexes with much lower frequency than inselected cells. Lesions in nonselected cells are therefore predominantlyunshielded and remain accessible to the cellular DNA repair machinery.As a result, genomic lesions in nonselected cells are repaired. Lesionrepair contributes to the survival of the nonselected cells. That is,the present compounds are relatively less genotoxic to nonselectedcells. As a result of the present method, the heterogenous cellpopulation becomes depleted of selected cells. Embodiments of thepresent method wherein the selected cell component that is sequesteredat the lesion site is a transcription factor are referred to as"transcription factor hijacking". In such embodiments, hijacking orsequestration of the transcription factor by the second agent at sitesother than the factor's natural genomic binding site further contributesto the death of selected cells, by inducing disarray in one or more ofthe cell's metabolic or secretory functions.

An advantage of the invention described herein is thatheterobifunctional compounds can be engineered that are selectivelyfatal (genotoxic) to a great phenotypic and phylogenetic variety ofselected cells. The term, "programmable genotoxic drugs" thus aptly sumsup the flexibility and adaptability of the inventive concept disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention, as well as the invention itself, will be more fullyunderstood from the following description of preferred embodiments, whenread together with the accompanying drawings, in which:

FIG. 1 is a schematic illustration showing basic features of theheterobifunctional programmable genotoxic compounds of the presentinvention and their mode of action as mediating steric hinderance of therepair of genomic lesions.

FIGS. 2A-2D present autoradiograph results of Southwestern (2A, 2B and2C) and Western Blot (2D) studies of the binding of an HMG-boxtranscription factor, hUBF (human UBF), to a structural decoy comprisinga cisplatin 1,2-dinucleotide intrastrand DNA adduct. (WCE=whole cellextract; hUBF obtained from in vitro translation; Anti-NOR-90=antiserumagainst hUBF.)

FIGS. 3A-3B present autoradiograph results of DNase I footprintingstudies showing that hUBF protects a region of the decoy symmetricallyspanning the cisplatin adduct site SEQ ID No: 3.

FIG. 4, top and bottom panels, presents autoradiograph results ofstudies establishing the affinity of hUBF for its endogenous genomicbinding site.

FIG. 5 presents autoradiograph results of additional footprintingstudies that revealed similarity in the hUBF-protected regions of thistranscription factor's cognate genomic binding site and the cisplatindecoy (compare FIG. 3).

FIGS. 6A-6B present autoradiograph results demonstrating binding ofstreptavidin to U-17 monoadducted with a heterobifunctional TMP-biotinconjugate. FIG. 6A presents an autoradiograph of the results of a gelmobility shift assay. 3200 cpm (.sup.˜ 0.1 nM) of the radiolabeledTMP-biotin lesioned DNA was used in each lane. 0 nM, 0.4 nM, 1 nM, 2 nM,5 nM, 10 nM, 50 nM and 50 nM of streptavidin were used in lanes 1 to 8respectively. In lane 8, free d-biotin was also added to the finalconcentration of 0.4 nM. FIG. 6B is a binding curve created by plottingthe percentages of bound probe (FIG. 6A) against streptavidinconcentrations.

FIG. 7 presents autoradiograph results demonstrating inhibition ofuracil gylcosylase by lesion-bound streptavidin. Bands (a) represent thefull-length and intact probe used in each reaction. Bands (c) representthe products of uracil glycosylase treatments and the subsequentpiperidine cleavages. Bands (b) and bands (d) are the breakdown productsof bands (a) and bands (c) respectively due to the alkali liability ofthe adducts.

FIG. 8 presents autoradiograph results of a DNase I protection assay.5000 cpm (.sup.˜ 1.5 fmoles, .sup.˜ 0.15 nM) of the ³² P end-labeled,TMP-biotin lesioned probe was used in each lane. 0 nM, 0.4 nM, 2 nM, 10nM, 50 nM and 50 nM of stretavidin were used in lanes 2 to 7respectively. In lane 7, 0.4 nM of free d-biotin was also included inthe incubation. Where indicated, 5 ug of DNase I (final concentration,0.4 mg/ml) was used in each digestion. The boxed thymidine base shown atright marks the position of the TMP-biotin monoadduct SEQ ID No. 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Broadly, the selectively genotoxic compounds disclosed herein comprise afirst agent that inflicts genomic lesions on cellular DNA, linked to asecond agent that attracts or sequesters at the genomic lesion site, asterically large cell component preferentially present in selected cellsof a heterogenous cell population. The cell component is stericallylarge enough to effectively hinder access to the lesion site by elementsof the cell's enzymatic DNA repair machinery, thereby shielding thelesion from repair. In preferred embodiments, the cell component is aprotein, such as a cell cycle control factor, a transcription factor, anoncogene product or a mutant tumor suppressor gene product, that isnormally engaged in the control of one or more genes relevant to thecell's growth or survival, or to secretory processes carried out by theselected cell.

FIG. 1 illustrates the basic principle of repair shielding by theheterobifunctional "programmable genotoxic" compounds of the presentinvention. A heterobifunctional compound 3 of the present invention isshown bound to cellular DNA of a nonselected cell 1 or of a selectedcell 2. In each cell, binding of the compound to cellular DNA results ina potentially repairable genomic lesion. The compound 3 comprises afirst agent 5 that binds to cellular DNA, linked, optionally by linker7, to a second agent 9 that binds to a cell component 12 preferentiallypresent in selected cells of a heterogenous cell population comprisingselected and nonselected cells. If unrepaired, the genomic lesioncontributes to the destruction of cells. Lesion repair is carried out bythe cell's enzymatic DNA repair machinery, which includes one or moresterically large repair enzymes 10. In the absence of the cell component12, repair enzymes 10 access and repair the lesion. However, in selectedcells, the cell component 12 binds to the second agent 9, effectivelyshielding the lesion from repair by presenting a steric obstacle torepair enzyme 10 access.

Genotoxic Agents useful as First Agents

The present compounds employ as first agent 5, genotoxic drugs that areknown in the art and can readily be prepared according to publishedtechniques, or are commercially available. Many of these genotoxic drugscurrently are used to treat infections and neoplastic diseases inmammals, e.g., humans. Analogs or derivatives of these drugs readily canbe prepared that are suitable for linkage to cell component bindingsecond agent 9 to obtain heterobifunctional genotoxic compound 3 of thepresent invention. Two general classes of compounds that are suitablefor use as first agent 5 are DNA alkylating agents and DNA intercalatingagents. Optionally, the first agent can be a precursor that becomesreactive with cellular DNA spontaneously or following exposure to anactivating stimulus, such as a cellular or secreted enzyme, cellmetabolite or metabolic byproduct, ionizing or nonionizing radiation,light energy, etc. For example, the first agent can be photoactivated.One class of photoactivatable first agents is represented by the drugpsoralen, a tricyclic furocoumarin that produces pyrimidine base adductsand crosslinks in cellular DNA. Tricyclic furocoumarin analogs andderivatives of psoralen can also be used as first agents. Thus, forexample, trimethylpsoralen (TMP) can be used herein. Psoralens are knownto be useful in the photochemotherapeutic treatment of cutaneousdiseases such as psoriasis, vitiligo, fungal infections and cutaneous Tcell lymphoma. Harrison's Principles of Internal Medicine, Part 2Cardinal Manifestations of Disease, Ch. 60 (12th ed. 1991). Anotherclass of photoactivatable first agents is represented by dacarbazine andincludes analogs and derivatives thereof.

Another general class of first agents, members of which can alkylate orintercalate into DNA, includes synthetically and naturally sourcedantibiotics. Of particular interest herein are antineoplasticantibiotics, which include but are not limited to the following classesof compounds represented by: amsacrine; actinomycin A, C, D(alternatively known as dactinomycin) or F (alternatively KS4);azaserine; bleomycin; carminomycin (carubicin), daunomycin(daunorubicin), or 14-hydroxydaunomycin (adriamycin or doxorubicin);mitomycin A, B or C; mitoxantrone; plicamycin (mithramycin); and thelike. Each class of antineoplastic antibiotics includes analogs andderivatives of the foregoing representative compounds. Antineoplasticantibiotics are known to be useful in the treatment of a variety ofneoplasms and viral diseases. Neoplasias currently manageable by theforegoing include leukemias, lymphomas, myelomas, neuroblastomas,neoplasias of bladder, testicular, endometrial, gastric, or lung origin,and others listed in Tables 301-6 and 301-7 of Harrison's Principles ofInternal Medicine, Part II Hematology and Oncology (12th ed. 1991). Agiven neoplasm is "manageable" by a given drug if treatment with thedrug alone or in combination with another drug confers some clinicallyrecognized benefit on the afflicted individual. Optimally, a partial ortotal remission is achieved. Drugs that contribute to a stabilization ofthe individual's clinical status or slow the progress of disease,however, are also considered beneficial and are used in the managementof neoplasias.

Still another general class of first agents, members of which alkylateDNA, includes the haloethylnitrosoureas, especially thechloroethylnitrosoureas. Representative members of this broad classinclude carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine and streptozotocin. Haloethylnitrosourea first agents can beanalogs or derivatives of any of the foregoing representative compounds.Neoplasias currently manageable by the foregoing include Hodgkin's,non-Hodgkin's and Burkitt's lymphomas, myelomas, glioblastomas andmedulloblastomas, pancreatic islet cell carcinomas, small cell lungcarcinomas and the like. Id.

Yet another general class of first agents, members of which alkylateDNA, includes the sulfur and nitrogen mustards. These compounds damageDNA primarily by forming covalent adducts at the N7 atom of guanine.Representative members of this broad class include chlorambucil,cyclophosphamide, ifosfamide, melphalan, mechloroethamine, novembicin,trofosfamide and the like. Nitrogen mustard or sulfur mustard firstagents can be analogs or derivatives of any of the foregoingrepresentative compounds. Nitrogen mustards are generally understood tocomprise the moiety N(CH₂ CH₂ X)₂, wherein X is a halogen, preferablychlorine. In mechloroethamine oxide, X is chlorine, and the moiety iscovalently bonded to a methyl (CH₃) group. Typically, then, nitrogenmustards such as chorambucil and mephalen have two reactive groups thatcan form covalent bonds with the N7 atoms of guanine residues. Thus,these drugs can form intrastrand or interstrand DNA crosslinks, and cancrosslink DNA to nucleophilic atoms in proteins. Each type of genomiclesion is thought to contribute to the lethal effects of nitrogenmustards. Neoplasias currently manageable by the foregoing includeHodgkin's, non-Hodgkin's, Burkitt's and other lymphomas, leukemias,myelomas, medullomas, neuroblastomas, small cell lung carcinoma,osteogenic sarcoma, neoplasias of breast, endometrial and testiculartissue, and the like. Id. U.S. Pat. No. 3,299,104 (issued Jan. 17, 1967)and Niculescu-Duvaz et al. (1967), J. Ned. Chem. 172-174, discloseestrogen, progesterone, androgen and steroid conjugates ofmechloroethamine. Muntzing and Nilsson (1972), 77 J. Krebsforch.166-170, report histologic studies conducted on cells of patientsreceiving one such conjugated methochloroethamine compound.

Yet a further general class of first agents, members of which formcovalent DNA adducts, includes heavy metal coordination compounds,including platinum compounds. Generally, these heavy metal compoundsbind covalently to DNA to form, in pertinent part, cis-1,2-intrastranddinucleotide adducts. Generally, this class is represented bycis-diamminedichloroplatinum(II) (cisplatin), and includescis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin),cis-diammino-(1,2-cyclohexyl)dichloroplatinum(II), andcis-(1,2-ethylenediammine)dichloroplatinum(II). Platinum first agentsinclude analogs or derivatives of any of the foregoing representativecompounds. Neoplasias currently manageable by platinum coordinationcompounds include testicular, endometrial, cervical, gastric, squamouscell, adrenocortical and small cell lung carcinomas along withmedulloblastomas and neuroblastomas. trans-Diamminedichloroplatinum (II)(trans-DDP) is clinically useless owing, it is thought, to the rapidrepair of its DNA adducts. The use of trans-DDP as a first agent hereinlikely would provide a compound with low toxicity in nonselected cells,and high relative toxicity in selected cells.

Other classes of first agents, members of which alkylate DNA, includethe ethylenimines and methylmelamines. These classes include altretamine(hexamethylmelamine), triethylenephosphoramide (TEPA),triethylenethiophosphoramide (ThioTEPA) and triethylenemelamine.Additional classes of DNA alkylating first agents include the alkylsulfonates, represented by busulfan; the azinidines, represented bybenzodepa; and others, represented by, e.g., mitoguazone, mitoxantroneand procarbazine. Each of these classes includes analogs and derivativesof the respective representative compounds.

Oligonucleotides or analogs thereof (e.g., phosphorothioateoligonucleotides, oligonucleotides incorporating O⁶ -methylguanineand/or O⁴ -methylguanine and the like) that interact covalently ornoncovalently with specific sequences in the genome of selected cellscan also be used as first agents, if it is desired to select one or morepredefined genomic targets as the locus of a genomic lesion. Suitableoligonucleotides that intercalate nonclassically into cellular DNA toform triple helices or other complex structures are disclosed in Riordanand Martin (1991), 350 Nature 442-443, the teachings of which areincorporated by reference. These compounds are expected to be useful forthe management of neoplasias whose growth characteristics are traceableto the aberrant activation of particular genes, such as cyclin genes,oncogenes and mutant tumor suppressor genes.

Each of the foregoing classes of suitable first agents comprises analogsand derivatives of the representative compounds mentioned herein. Ananalog of a representative compound can be a structurally relatedcompound, optionally a precursor of a representative compound or aderivative of a precursor. For example, trimethylpsoralen is an analogof the representative compound psoralen. An analog can also be acompound bearing substituents that are structurally and/or functionallyanalogous to those of a representative compound. For example, if arepresentative compound has a chlorine substituent, an analog can haveanother halogen substituent (e.g., bromine or fluorine). A derivative ofa representative compound is chemically, physicochemically ormetabolically synthesized from a representative compound, and cancomprise a greater or lesser number and complexity of substitutents thanthe representative compound. Appropriate substituents to the basicstructure of the representative compounds in each class will be known orcan be determined through no more than routine experimentation orcomparative inspection of the structures of two or more representativemembers of a particular class. Substitutents suitable for use in thevarious classes of first agents listed above thus include linear,branched or cyclic alkyl, aryl or mixed alkyl and aryl groups; organicor inorganic acids, bases or neutral moieties. Substituents present inanalogs and derivatives of the representative compound can modulate DNAbinding activity (e.g., enhance or impair activity), but should notabrogate such activity.

Preferred Classes of Cell Components Bound by Second Agent 9

Turning to second agent 9 of heterobifunctional compound 3, it should benoted that, in all embodiments, the second agent serves to mediateattachment of a cellular macromolecule to the site of genomic damagecaused by binding of the first agent 5 to cellular DNA. The boundcellular macromolecule sterically shields the lesion from repair. Thesecond agent either binds directly to the cellular macromolecule, or toa ligand, cofactor or metabolite to which the cellular macromolecule inturn binds with high affinity. In either circumstance, the second agentmediates formation of a stable complex between the cellularmacromolecule and the heterobifunctional compound. As the complex isstable under intracellular, e.g., nuclear, conditions, it acts as apersistent steric shield, preventing repair of the genomic lesion for asufficiently long period of time for the lesion to contribute to thedemise of selected cells.

In preferred embodiments, the second agent binds directly to a cellcomponent that is a macromolecule, such as a protein, preferentiallyassociated with selected cells in a heterogenous cell population. Thisprotein provides a phenotypic distinction between selected andnonselected cells of the population. It can be a protein of endogenouscellular origin (expressed from the cellular genome), or of viral origin(expressed from the genome of a virus infecting the selected cells).Selected cells are phenotypically distinguished from nonselected cellsby the qualitative or quantitative association of the secondagent-recognized protein. Thus, nonselected cells lack the protein, orare associated with diminished amounts thereof. The recognized proteincan be a phylogenetic species or tissue-type variant of a correspondingprotein associated with nonselected cells. It can be a protein, theexpression of which is developmentally regulated or dysregulated inselected cells in a manner different from its regulation in nonselectedcells. It can also be a mutant of a protein normally associated withnonselected cells. Examples of recognized proteins thus includebacterial, fungal, parasitic and viral intracellular proteins. Otherexamples include developmental stage specific proteins, includingproteins expressed upon dedifferentiation or malignant transformation ofnonselected cells into selected cells. Still other examples includeproteins preferentially expressed by dividing cells, or proteins thatare relevant to the process of cell division (cell cycling). Otherexamples include proteins that can be induced in selected cells byirradiation or other stimuli to which selected cells respond.

Selected cells with which the second-agent recognized protein isassociated, therefore, can be dividing cells, e.g., transformed cells.Preferably, selected cells have at least about a 5-fold excess of therecognized protein, over the amount of the same or a correspondingprotein in nonselected cells. More preferably, the excess is at leastabout 10-fold. Still more preferably, the excess exceeds about 100-fold.Even more preferably, the recognized protein is undetectable innonselected cells. In many preferred embodiments, the recognized proteinis intracellular. For example, the recognized protein is a nuclearprotein or is a protein that is normally translocated to the nucleus,e.g., when bound to a transport protein or an activating or suppressingligand. Preferred classes of second agent-recognized intracellularproteins therefore include but are not limited to cyclins, cyclindependent kinases, ocogene products, mutant tumor suppressor geneproducts, and transcription factors.

Oncogenes are genes encoding proteins that are relevant to the processof malignant transformation of a normal cell into a malignant(cancerous) cell. Thus, oncogenes encode proteins relevant to theproliferation and differentiation states of a cell. Molecular CellBiology, Ch. 24 Cancer, 967 and 984-994 (2d ed. 1990). Oncogenes can befound in the cellular genome, or in the genome of a virus infecting thecell. Infection with certain tumorigenic viruses causes the infectedcell to undergo malignant transformation. Examples of such virusesinclude the adenoviruses and papovaviruses (e.g., SV40 and polyoma),retroviruses (e.g., Rous sarcoma virus, mouse mammary tumor virus, humanT-cell leukemia virus-1, Epstein-Barr virus, and the papilloma viruses).Id., Ch. 24 Cancer, 967-980. Oncogenes encoding intracellular proteins(e.g., src, yes, fps, abl, met, mos and crk), particularly thoseencoding nuclear proteins (e.g., erbA, abl, jun, fos, myc, N-myc, myb,ski and rel) are of particular interest herein. Id. at Table 24-1. Thatis, certain preferred second agents 9 bind to nuclear oncogene products.Oncogenes of both viral and cellular origin have been implicated in theetiology of numerous neoplasias. Harrison's Principles of InternalMedicine, Part 1 Biological Basis of Disease, Ch. 10 (12th ed. 1991).These include, but are not limited to Burkitt's lymphoma (Epstein-Barrvirus; activation of endogenous myc), chronic myelogenous leukemia(activation of abl), anogenital cancers (papilloma viruses), andpancreatic carcinomas (ras). Id. at Table 10-3.

Tumor suppressor genes are also known as "anti-oncogenes" or "repressiveoncogenes" because they encode proteins (gene products) thataffirmatively maintain cells in an appropriately differentiated state,and/or restrain cells from embarking on unbridled rounds ofproliferation. These desirable properties can be lost upon mutation of atumor suppressor gene, freeing the cell from normal growth controls.Tumor suppressor gene products are thought to bind either to cellularDNA (and thus may themselves be transcription factors), or to otherproteins, e.g., oncogene products. Two examples of tumor suppressorgenes are Rb, the retinoblastoma gene (Molecular Cell Biology, Ch. 24Cancer, 996 (2d ed. 1990); Harrison's Principles of Internal Medicine,Part 1 Biological Basis of Disease, Ch. 10, 68-69) and the nuclearphosphoprotein p53 (Hollstein et al. (1991), 253 Science 49-53). p53 isof particular interest herein, as somatic mutations of p53 have beenreported in sporadic and inherited neoplasms of breast, colon, lung,esophagus, liver, brain, blood-forming (myeloid and lymphoid),reticuloendothelial and other tissues (Id.). Indeed, somatic mutationsof p53 are thought to play a role in up to one-half of all newmalignancies documented yearly in Britain and the United States, makingthis protein the most frequent target for mutation in human cancers(Vogelstein (1990), 348 Nature 681-682; Marx (1990), 250 Science 1209).Studies investigating germ-line mutations of p53 in familial Li-Fraumenisyndrome, an inherited susceptibility to cancers associated with p53mutation, have shown that small deletions, transpositions and pointmutations affecting conserved regions of the protein convert p53 from asuppressive growth regulatory protein into a transdominant oncogene,which can bind to and inactivate wildtype p53 (Gannon et al. (1990), 9EMBO J. 1595-1602; Malkin et al. (1990), 250 Science 1233-1238; andSrivastava et al. (1990), 348 Nature 747-749). Second agents 9 whichbind to mutant, but not wildtype, p53 are accordingly preferred incertain embodiments of the present invention. The precise nature andlocations of transforming mutations in p53 has been the subject ofintense investigation, and is reviewed in Hollstein et al. (1991), 253Science 49-53, the teachings of which are herein incorporated byreference. At least one monoclonal antibody, PAb240, that recognizesmutant but not wildtype p53 has been isolated, and the recognizedepitope characterized (Gannon et al. (1990), 9 EMBO J. 1595-1603;Stephen and Lane (1992), J. Mol. Biol. 577-580; the teachings of both ofwhich are incorporated herein by reference). Second agents 9 that bindthe epitope recognized by PAb240 are particularly preferred in certainembodiments of the invention.

Cyclins, cyclin dependent kinases, and cyclin associated proteinstogether form nuclear complexes that control initiation and progressthrough the cell cycle. Keyomarsi and Pardee (1993), 90 Proc. Natl.Acad. Sci. USA 1112-1116, and Xiong et al. (1993), 7 Genes and Dev.1572-1583, the teachings of each of which are incorporated herein byreference. Cyclins and cyclin dependent kinases are classified,according to the presence therein of conserved amino acid sequencemotifs, as members of evolutionarily conserved multigene families thatdetermine and regulate cell proliferation. The particular cyclins andcyclin dependent kinases that are associated in nuclear cell cyclecontrol complexes shift subtly at different stages of the cell cycle(e.g., upon transition from G₁ to S or upon transition from G₂ to M).Xiong et al. report that cyclin expression and associated proteins arederanged in transfomed cells. Thus, as for tumor suppressor geneproducts, malignant transfomation may be associated with theinappropriate display of a cryptic epitope in one or more cyclins,cyclin dependent kinases or cyclin associated proteins. Such a crypticepitope might prevent normal association between cyclins and cyclindependent kinases, or might promote inappropriate associations. Incertain embodiments, then, second agents 9 of the presentheterobifunctional compounds bind selectively to such crypticcyclin-related epitopes. Keyomarsi and Pardee report that one cyclin,Cyclin E, is significantly overexpressed in breast carcinoma cells,relative to its expression level in normal breast tissue. Accordingly,the accumulation of cyclin E offers a phenotypic distinction betweenselected (transformed) and nonselected (normal) cells in breast tissue.In certain embodiments, second agents 9 of the present invention thatbind to Cyclin E thus offer the ability to selectively destroy breastcarcinoma cells.

Transcription factors are proteins that bind to specific sites incellular DNA (e.g., specific sequences, structures or a combinationthereof) and thereby regulate the expression of one or more genes. Suchsites in the cellular DNA are referred to herein as endogenous genomicbinding sites. Transcription factors can, by binding to their cognateendogenous genomic binding sites, promote, enhance or repress geneexpression. Molecular Cell Biology, Ch. 11 Gene Control and Developmentin Eukaryotes, 400-412 (2d ed. 1990). Transcription factors can begrouped into the following classes, based upon similarities in proteinstructure in the regions thought to interact with DNA: helix-turn-helixor homeobox proteins; zinc-finger proteins; and amphipathic helicalproteins, such as leucine-zipper proteins. Second agents can thus bedesigned according to the principles set forth herein to bind to one ormore structurally similar transcription factors. In some embodiments,second agents mimic endogenous genomic binding sites for particulartranscription factors. These binding site mimics are referred to hereinas transcription factor decoys. Binding affinity of a giventranscription factor for a decoy is preferably near the affinity of thefactor for its endogenous genomic binding site. That is, bindingaffinity of the factor for the decoy is within about 100-fold of itsaffinity for the cognate site (e.g., if K_(d)(app) for the cognate siteis 1 nM, the K_(d)(app) for the decoy is about 100 nM). Preferably,affinity of the decoy is within about 10 fold that of the cognate site.More preferably, affinity of the decoy exceeds that of the cognate site.Particular transcription factor decoys that mimic the sequences ofendogenous genomic binding sites for particular transcription factorsare disclosed in Bielinska et al. (1990), 250 Science 997-1000, and inChu and Orgel (1992), 20 Nucl. Acids Res. 5857-5858, the teachings ofeach of which are incorporated herein by reference. The presentinvention extends these teachings to encompass transcription factordecoys that mimic the structures of endogenous genomic binding sites fortranscription factors, including structures that aresequence-independent.

In appropriate embodiments, second agents 9 are employed that are decoysfor transcription factors that control the expression of genes relevantto the growth or survival of selected cells, or to metabolic orsecretory processes carried out by selected cells. Exposure of selectedcells to such compounds results in transcription factor hijacking. Thatis, a transcription factor bound to the decoy is titrated away from itsnatural genomic binding site and becomes sequestered at the site of agenomic lesion. Preliminary studies were carried out to confirm that avital transcription factor could be "hijacked" in this manner and causedto bind to a genomic lesion. These preliminary studies demonstrated thatan HMG box transcription factor, upstream binding factor (UBF), binds tocisplatin 1,2-d(GpG) intrastrand crosslinks (G G) and to its naturalgenomic binding site with comparable affinities. For these studies,human upstream binding factor (hUBF) was used. hUBF binds to theupstream control element or UCE of the human ribosomal RNA (rRNA)promoter and is an important positive regulator of rRNA transcription(Jantzen et al. (1990), 344 Nature 830-836). rRNAs are required elementsof the cellular protein synthesis machinery. Thus, hUBF/promoter bindingis relevant to the proper functioning of the cell's protein synthesismachinery.

It should be noted that the particular nucleic acid used as an hUBFdecoy in the preliminary studies lacked sequence similarity to thesequence of the endogenous genomic binding site for hUBF; thus,transcription factor hijacking was accomplished by structural, ratherthan sequence-specific, recognition. Southwestern and Western blotanalysis yielded results, presented in FIG. 2, showing that in vitrotranslated hUBF bound to DNA globally modified by cisplatin but failedto recognize unmodified DNA or DNA containing adducts of thegenotoxically inactive isomer, trans-DDP. Proteins in crude HeLa cellextracts having molecular weights that correlate to the known sizes ofhUBF species displayed a similar binding preference. DNase Ifootprinting analysis (FIG. 3) of the cisplatin-modified nucleic aciddecoy showed that hUBF protected the 14 bp DNA region that symetricallyflanks the site of a defined cisplatin G G adduct (Panel A, Lane 1).These results directly demonstrate that hUBF binding prevented access tothe cisplatin lesion site by a sterically large DNA processing enzyme.Competition studies (results presented in FIG. 5) established that thecisplatin decoy efficiently inhibited the formation of complexes. Thatis, the structural decoy was shown to be an effective competitiveinhibitor of the proper binding of hUBF to its cognate, sequencespecific, genomic binding site (the UCE). The affinity of hUBF for G Gwas substantial (K_(d)(app) =60 pM; see FIG. 3, Panel B). Forcomparison, the K_(d)(app) of another HMG box protein, HMG1, for thecisplatin G G adduct has been shown to be 370 nM (Pil and Lippard(1992), 256 Science 234-237). FIG. 5 also reveals a significantnonspecific binding component of hUBF for its promoter. This is alsoobserved with other HNG box proteins, including lymphoid enhancerfactor-1 (LEF-1), which binds with nominal specificity (20-40 fold) toits putative genomic recognition sequence (Giese et al. (1991), 5 Genes& Dev. 2567-2578). Footprinting studies (results presented in FIG. 5)also established similarity between the hUBF protected regions of thecognate genomic binding sequence (the UCE) and the cisplatin decoy. Fromthese results, it can be predicted that the levels of cisplatin thataccumulate in cellular DNA in vivo upon treatment of a cancer patientwith a cisplatin chemotherapeutic regime are sufficient to titrate hUBFaway from the ribosomal RNA promoter.

The above-summarized preliminary studies lend insight into thestructural features of hUBF-cisplatin decoy complexes. The cisplatinadduct was approximately centered within the 14 bp protected region(FIG. 3), suggesting that the DNA binding domain(s) is symmetricallyplaced relative to the adduct. As discussed hereinabove, cisplatinadducts cause structural distortions in duplex DNA. 1,2-Dinucleotideadducts are bent and partially unwound in the area immediatelyassociated with the platinum coordination complex. The resulting angularstructure thus has an "elbow" at the lesion site. This elbow appears toremain solvent exposed, even when the lesion is shielded by bound hUBF:the phosphodiester bond immediately 5' to the lesion remained sensitiveto DNase I. This result is consistent with binding of hUBF to the minorgroove of duplex DNA, on the convex side of the DNA bend. Others havereported similar findings concerning the binding of other HHG boxtranscription factors, particularly LEF-1 and SRY (the testisdetermining factor) to their cognate genomic binding sites through minorgroove interactions (Giese et al. (1991), 5 Genes & Dev. 2567-2578; vande Wetering and Clevers (1992), 11 EMBO J. 3039-3044; King and Weiss(1993), 90 Proc. Natl. Acad. Sci. U.S.A. 11990-11994).

hUBF, like SRY, exhibits both sequence-specific and structure-specificmodes of DNA recognition. The footprinting data suggest that thestructure-specific hUBF-G G decoy! and sequence-specific hUBF-UCE!complexes share structural features. In each case, a protected regionsymmetrically flanks a nuclease sensitive site. DNA bending is thelikely common feature of these complexes. Indeed, a hallmark of the HMGdomain is its propensity to interact with bent DNA and also to inducebending in linear sequences. SRY, to give one example, efficientlyrecognizes four way DNA junctions with sharp angles (Ferrari et al.(1992), 11 EMBO J. 4497-4506). Furthermore, SRY induces a sharp bend(85°) in a specific DNA sequence upon binding (Giese et al. (1992), 69Cell 185-195). The specific interactions of the HMG domain with bentDNAs may be attributed to its "L" shaped cleft, as reported recently(Weir et al. (1993), 12 EMBO J. 1311-1319). hUBF probably also bendsDNA, although detailed structural studies have yet to be performed. TheDNase I hypersensitive site induced in the UCE upon hUBF binding mayindicate DNA bending because DNase I activity is sensitive to structuralfeatures of DNA, including the width of the minor groove (Drew andTravers (1984), 37 Cell 491-502). The putative bend site is centeredwithin a UCE region that is protected from DNase I; interestingly, the GG-induced DNA bend is also centered within a DNase I-resistant region.Thus, it appears that the bent and unwound DNA structure induced by G Gmimics a favorable DNA conformation that occurs during the formation ofa stable hUBF-rDNA promoter! complex. A similar model was proposedrecently to explain structure-specific recognition by SRY (King andWeiss (1993), 90 Proc. Natl. Acad. Sci. U.S.A. 11990-11994).

Results of in vitro competition assays (shown in FIG. 5) furtherestablished that the cisplatin decoy interacts with hUBF by substitutingfor the transcription factor's endogenous genomic binding site (the UCE)in the rDNA promoter. By logical implication, introduction of cisplatindecoys into the cellular milieu is expected to hijack hUBF and inducedisarray of cellular processes normally dependent on properhUBF-promoter! complexing. In particular, the formation of high affinityhUBF-decoy! complexes should reduce the amount of hUBF available forpromoter binding. The steep relationship between promoter occupancy andnuclear hUBF concentration (FIG. 4) indicates that even a small degreeof sequestration of hUBF by cisplatin lesions can significantly impairexpression of nucleolar genes encoding rRNAs. Thus, rRNA transcription,and therefore cellular protein synthesis, will be compromised.Furthermore, binding of a sterically large protein, such as hUBF, tocisplatin lesions impedes or inhibits DNA repair. Indeed, studies haveshown that, although G G adducts are excised from cellular DNA in humancells (Fichtinger-Schepman et al. (1987), 47 Cancer Res. 3000-3004),this repair process is inefficient (Szymkowski et al. (1992), 89 Proc.Natl. Acad. Sci. U.S.A. 10772-10776). Results presented herein showedthat the 14 bp region symetrically flanking the G G lesion in thecisplatin decoy was strongly protected from nuclease cleavage. Fromthis, it is reasonable to predict that this region would also beshielded from components of the enzymatic DNA repair machinery. Infurther support of this prediction, the XPAC protein, which recognizesdamaged DNA and is essential for human nucleotide excision repair, has arelatively low affinity for G G cisplatin lesions (K_(d)(app) >600 nM)(Jones and Wood (1993), 32 Biochemistry 12096-12104). XPAC, therefore,should not displace hUBF, which binds much more avidly to cisplatinlesions. hUBF therefore acts as an effective shield protecting cisplatingenomic lesions from repair.

Both DNA repair and protein synthesis are likely to be more critical forproliferating cells, such as those of tumors, than for quiescent cells,such as those of normal differentiated tissue (Mauck and Green (1973),70 Proc. Natl. Acad. Sci. U.S.A. 2819-2822; Fraval and Roberts (1979),39 Cancer Res. 1793-1797). The numbers of intracellular hUBF molecules,and of cisplatin genomic lesions formed in a typical round ofchemotherapy, have been calculated. Both are in the range of about 5×10⁴/cell (Bell et al. (1988), 241 Science 1192-1197; Reed et al. (1993), 53Cancer Res. 3694-3699). Biologically significant and synergisticassaults on the viability of selected cells should therefore follow fromthe cisplatin-hUBF interactions predicted by both the hijacking andshielding models for cisplatin genotoxicity.

Still other classes of proteins for which second agents 9 of the presentinvention can be designed comprise nucleic acid processing proteins,e.g., ribonucleic acid (RNA) processing proteins, including proteinsinvolved in splicing RNA gene transcripts to produce messenger RNA(mRNA). In addition, second agents can be designed that bind to othercellular macromolecules, e.g., RNA transcripts or portions thereof ofgenes that are actively expressed in selected cells but not innonselected cells.

Cell-Component Binding Compounds Useful as Second Agent 9

Turning now to the structural features of second agent 9, it has beendisclosed above that the second agent can be a ligand or an analog orderivative thereof, that is bound by the above-discussed proteinpreferentially associated with selected cells. Such ligands includeestrogens, progesterones, androgens, glucocorticoids and other solublehormones, toxins, clinically useful analogs, and metabolites, both ofintracellular and extracellular origin. Heterobifuntionalligand-genotoxic agent compounds have been prepared and subjected topreliminary studies that support extension of the cisplatin-based lesionshielding concept according to the first model described above. In onesuch compound, the ligand biotin was linked, through the use of standardtechniques, to a genotoxic first agent. The particular first agentselected was the photoactivatable drug trimethylpsoralen (THP), but itshould be understood that any of the first agents disclosed herein couldhave been used. Psoralen compounds intercalate into double-stranded DNAat d(TpA) dinculeotides and form mono and diadducts therewith uponexposure to near-UV irradiation (Cimino et al. (1985), 54 Ann. Rev.Biochem. 1151-1193). Biotin binds with extraordinarily high affinity tothe proteins avidin and strepatavidin (K_(d)(app) 10⁻¹⁵ M, Green (1975),25 Adv. Protein Chem. 85-133) and thus is widely used in research andclinical assays, such as enzyme-linked immunosorbent assays (ELISA),that capitalize on specific protein interactions to detect or quantitatea protein of interest. In this study, the biotin-THP compound was mixedwith duplex DNA comprising an additional, defined genomic lesion(deoxyuridine, the deamination product of cytosine) located in theimmediate vicinity (within three base pairs) of the TMP lesion site. Asdescribed more fully in the Examples, the biotin-TMP compoundeffectively bound concurrently to duplex DNA and streptavidin, althoughboth binding affinities were lower in the heterobifunctional compoundthan in the unconjugated precursors of the first and second agents.Quantitative gel shift assays revealed a K_(d) of .sup.˜ 1.5 nM betweenstreptavidin and the immobilized biotin. This K_(d) value differssignificantly from that of free biotin for streptavidin or avidin (theK_(d) for free biotin with streptavidin or avidin is about 10⁻⁶ nM).Assuming that the biotin domain in the TMP-biotin conjugate behavedsimilarly to free biotin, the significant increase of the K_(d) value(in other words, the decrease in binding affinity between biotin andstreptavidin) was probably caused by the covalent binding of TMP-biotinconjugates to DNA. This effect likely can be minimized by furtheroptimizing the linkage technique and nature of the linker employed.Techniques such as those described herein can be applied or adaptedthrough no more than routine experimentation to accomplish this goal. Asdescribed, an optimal distance between a second agent ligand and the DNAhelix should allow tight binding between the ligand and the cellcomponent and yet adequately shield the DNA region vicinal to the adductsite (i.e., the genomic lesion). Streptavidin, when bound to damaged DNAat genomic lesion sites, shielded adjacent deoxyuridine lesions fromrepair by the appropriate DNA repair enzyme, uracil gylcosylase. Theseresults are presented below in the Examples. The size of the shieldedregion, at least 20 adjacent nucleosides, was comparable to that of theDNA patch typically released by DNA excision repair enzymes.

Another heterobifunctional ligand-genotoxic agent compound, in this casean estradiol-chorambucil conjugate, has been prepared. This programmedgenotoxic compound should mediate adherence of intracellular estrogenreceptors to genomic lesions inflicted by chorambucil. Again, it shouldbe understood that, through appropriate standard techniques, theestrogen ligand could have been linked to any of the genotoxic firstagents disclosed herein. Guidance is presented herein for confirmingthat estrogen receptors also can be used to shield genomic lesionseffectively from repair by the cellular enzymatic DNA repair machinery,thereby contributing to the demise of selected cells that expressestrogen receptors. According to the principles of the presentinvention, heterobifunctional compounds programmed to recruit theestrogen receptor to become a shield for genomic lesions comprisegenotoxic first agent, an optional linker, and a second agent thataffixes the receptor protein to the site of a lesion in cellular DNAcaused by the genotoxic agent. Preferably, the genotoxic first agent isitself bifunctional and thus offers the capability of forming intra- orinterstrand crosslinks in cellular DNA. Linking groups of varying lengthand molecular composition allow the practitioner to optimize the presentcompounds for concurrent binding of estrogen receptors and DNA.

There is precedent indicating that estrogen ligands that are affixed tolarge carrier molecules or to a solid support such as agarose, can stillattract and bind the estrogen receptor from solution. The estrogenprecursor estradiol has been linked at either the 7α or 17α position toagarose, creating a means to isolate the estrogen receptor from cellextracts by affinity chromatography (Sica et al. (1973), 248 J. Biol.Chem. 6543-6558; Bucort et al. (1978), 253 J. Biol. Chem. 8221-8228;Redeuilh et al. (1980), 106 Eur. J. Biochem. 481-493). DNA and agaroseboth have polysaccharide character. Unlike agarose, however, DNA monomerunits of deoxyribose are linked by charged phosphodiester groups and arealso bonded to heterocyclic purines or pyrimidines containing bothnitrogen and oxygen atoms. Hydrogen atoms bonded to nitrogens andoxygens form hydrogen bonds within the helical DNA molecule, and canalso form such bonds with proteins and other diffusible molecules. Suchassociations could assist in the formation of a lesion-shielding complexbetween the cell component, the heterobifunctional compound and cellularDNA, by analogy to interactions between cellular DNA and nuclearproteins that determine DNA structure and regulate gene expression. Itis also possible, however, that hydrogen bonding could adversely affectthe ability of the estrogen ligand to bind its receptor. Optimization ofa linker disposed between the estrogen ligand and the genotoxic agentshould, however, project the ligand sufficiently away from the DNAmolecule, facilitating a high affinity interaction with the boundshielding protein.

Because appropriate precursors are readily available, the preparation of17α linked derivatives of estradiol are the simplest from a syntheticchemical viewpoint. For example, starting with estrone (3-hydroxy-1,3,510!-estriene-17-one), substitution of a short amino alcohol at the 17αposition of estradiol can be achieved using the Grignard reaction.Alternatively, 17α-ethynylestradiol can be used as a starting point forattachment of a short alkyl amine. The reported synthetic routes to 7αestradiol derivatives are more complex, but should still be within theabilities of those skilled in the art. Charpentier et al. (1988), 52Steroids 609-621, synthesized 7α-carboxymethyl-9(11)-ene derivatives ofestrone and estradiol starting with adrenosterone, in which acarboxymethyl group was first introduced at the 7α position and then theA ring was aromatized. In another published synthesis of 7α derivatives,Bucort et al. (1978), 253 J. Biol. Chem. 8221-8228 described theconjugate addition of a Grignard reagent to a canrenoate methylester, toultimately produce a 7α carboxylic acid derivative of estradiol.Alternatively, attachment of the steroid ligand through the amino groupat the end of the short alkyl chain can be accomplished by allowing anappropriately protected molecule to react with p-nitrophenylchloroformate.

From the examples presented below, one of skill can readily adapt themethods used to demonstrate function of the streptavidin-attractingheterobifunctional compound suitably for demonstrating the biochemicaland in vitro functionality of heterobifunctional compounds that comprisea ligand decoy for the estrogen receptor. Additional guidance isprovided in the prospective examples set forth below, particularly forassessing the ability of the ligand decoy to form interstrand crosslinksin DNA; assessing whether an appropriate lesion shielding complex isformed between damaged DNA and the estrogen receptor; and assessing thepresent ligand-decoy compound for selectively killing cells in vitrothat express the estrogen receptor. Similar techniques can be applied oradapted with no more than routine experimentation, to demonstratefunctional properties of heterobifunctional compounds programmed toattract other ligand-responsive transcription factors. Indeed, suchstudies are appropriate for evaluating the functionality of otherheterobifunctional compounds, such as compounds designed to attractoncogene products, tumor suppressor gene products, cyclins, and the likeand affix these cell components to the sites of genomic lesions.Electrophoretic mobility shift and DNase I protection analysis aresuitable techniques generally for demonstrating whether a particularheterobifunctional compound forms genotoxic lesions, whether a chosencell component is bound by a suitably programmed heterobifunctionalcompound, and whether the resulting complex is effective for shieldinggenomic lesions from the action of enzymes that act on cellular DNA.

As is apparent from the preliminary studies carried out with hUBF,second compound 9 can also be a nucleic acid that mimics an endogenousgenomic binding site of a transcription factor or other protein to besequestered at genomic lesion sites. Nucleic acid second agents can besingle stranded, double stranded, linear, branched, circular or acombination of these configurations. Either RNA or DNA can be used.Through intrastrand base pairing, linear or circular nucleic acid secondagents can adopt stable hairpin or dumbell configurations (Chu and Orgel(1992), 20 Nucl. Acids Res. 5857-5858). Certain second agents(transcription factor decoys) can resemble either the sequence or thestructure of the recognized transcription factor's endogenous bindingsite. That is, the nucleotide sequence of the decoy can comprise thesequence of the endogenous site, or a sequence sufficiently homologousthereto to confer protein-binding activity on the decoy. For example,the decoy sequence can be a conservative variant of the endogenoussequence. Preferably, the decoy sequence is more than 50% identical tothe endogenous sequence. More preferably, it is more than 70% identical,and even more preferably, it is more than 90% identical. It is wellknown that the binding avidity of many nucleic acid binding proteinsthat recognize specific nucleotide sequences can be enhanced by nucleicacid regions adjacent to the actual binding site. These flanking regionscan be disposed 3' or 5' to the specific, recognized sequence. As thenucleic acid binding protein need not interact directly or strongly withnucleotides in the flanking region, greater sequence variability can betolerated at such locations than in the binding site itself. Thus,decoys can be constructed that comprise a core, conserved bindingsequence flanked by adjacent regions that modulate, e.g., enhance,binding preference of the protein to the decoy relative to theendogenous site. Similar principles can be applied to the constructionof nucleic acid decoys that mimic nucleic acid structures rather thansequences. Structural features recognized by the protein can be producedby folding, looping, kinking, adoption of higher ordered structures(e.g., cruciforms) or of nonclassical helix configurations (e.g., ZDNA)by the nucleic acid decoy. Optionally, these structural features can bestabilized by non-nucleic acid components of the decoy, such ascrosslinking agents. Still further variation can be introduced, andfavorable properties (e.g., stability under in vivo conditions)emphasized, by the use of nucleotide analogs or derivatives such asphosphorothioate analogs, or O₆ - and/or O⁴ -methylguanine derivatives,in the decoy sequence.

An important class of nucleic acid second agents 9 includes those knownin the art as "aptamers". Aptamers are the products of directed, alsoknown as in vitro, molecular evolution. The term "aptamer" wasoriginally coined by Ellington and Szostak to describe the RNA productsof directed molecular evolution, a process in which a nucleic acidmolecule that binds with high affinity to a desired ligand is isolatedfrom large library of random DNA sequences (Ellington and Szostak(1990), 346 Nature 818-822). The process involves performing severaltandem iteratations of affinity separation, e.g., using a solid supportto which the desired ligand is bound, followed by polymerase chainreaction (PCR) to amplify ligand-eluted nucleic acids. Each round ofaffinity separation thus enriches the nucleic acid population formolecules that successfully bind the desired ligand. In this manner,Ellington and Szostak "educated" an initially random pool of RNAs toyield aptamers that specifically bound organic dye molecules such asCibacron Blue (Id. at FIG. 2). Certain of the aptamers obtained coulddiscriminate between Cibacron Blue and other dyes of similar structure,demonstrating specificity of the technique. Aptamers can even beengineered to distinguish between stereoisomers that differ only byoptical rotation at a single chiral center (Famulok and Szostak (1992),114 J. Am. Chem. Soc. 3990-3991). Originally, it was thought that RNAaptamers would be more suitable for ligand recognition, in view ofestablished knowledge of naturally occurring RNAs with higher orderedthree-dimensional structures (e.g., rRNA or transfer RNA, tRNA).However, single-stranded DNA molecules produced by asymmetric PCRamplification were also shown effective (Ellington and Szostak (1992),355 Nature 850-852). It should be noted that aptamers can be preparedfrom nucleotide analogs, such as phosphorothioate nucleotides, which canoffer increased aptamer stability under physiological conditions.Standard techniques are available for linking nucleic acids, such astranscription factor decoys and aptamers, to other chemical moieties,such as genotoxic drugs, without substantial loss of protein-recognitioncapability and genotoxicity.

The principles of directed molecular evolution encompass the productionof aptamers that bind with high affinity to proteins, such as DNAbinding proteins, including transcription factors (Tuerk and Gold(1990), 249 Science 505-510; Famulok and Szostak (1992), 31 Angew. Chem.Intl. Ed. Engl. 979-988, the teachings of which are herein incorporatedby reference). Recently, an aptamer has been reported that binds withhigh affinity to the extracellular protein thrombin (Bock et al. (1992),355 Nature 564-566), and can even inhibit thrombin catalyzed blood clotformation. High affinity aptamers can be generated even against proteinsfor which there is little or no structural or ligand-recognitioninformation available (Famulok and Szostak (1992), 31 Angew. Chem. Intl.Ed. Engl. 979-988; see discussion concerning the HIV Rev protein). Thus,aptamer second agents can be generated, through available techniques,that bind to virtually any desired selected-cell associated protein,whether or not the protein has a known natural ligand or endogenousgenomic binding site. This flexibility offers great promise in thedesign of programmable genotoxic drugs useful in selectively destroyingneoplastic or virally infected cells, such as cells infected with thehuman immunodeficiency virus (HIV) or tumorigenic adenoma and papillomaviruses. The aptamer-recognized protein can be a member of any of thegeneral classes discussed herein: transcription factors,ligand-responsive transcription factors, oncogene products, tumorsuppressor gene products, cell cycle regulatory proteins, nucleic acidprocessing proteins, nuclear structural proteins, and the like. Apreferred aptamer binds to the nuclear phosphoprotein p53. Aparticularly preferred aptamer binds to a region of tumor-associatedmutant p53 that is cryptic in wildtype p53, such as the PAb240 epitope(Gannon et al. (1990) 9 EMB0 J. 1595-1602; Steven and Lane (1992), 255J. Mol. Biol. 577-583). As described more fully in the examples, apopulation of aptamers that bind selectively to the PAb240 epitope hasbeen prepared. Heterobifunctional compounds comprising an aptameramplified from this pool and thus programmed to bind mutant p53 can beassessed for biomolecular and in vitro function through appropriateadaption of the techniques and guidelines set forth below in the actualand prospective examples. Such adaptions should be well within theabilities of those of skill in the art.

Yet another general class of second agents 9 includes peptide ligandsselected from so-called epitope libraries. Libraries of random peptidesof defined average length, and techniques for preparing such librariesare available. Such libraries have been used for determining the preciseepitope recognized by an antibody of interest (Geysen et al. (1984), 81Proc. Natl. Acad. Sci. USA 3998-4002; Fodor et al. (1991), 251 Science767-773, the teachings of each of which are incorporated herein byreference). At least one "living library" has been constructed, fromfilamentous bacteriophage expressing random peptide epitopes cloned intoa viral coat protein (Scott and Smith (1990), 249 Science 386-390, theteachings of which are incorporated herein by reference). Thistechnology offers the advantages that phage displaying a peptide withfavorable binding characteristics can be affinity purified against adesired protein component of selected cells (e.g., a transcriptionfactor, cyclin, intracellular receptor, or tumor suppressor geneproduct), propagated in vivo using a bacterial host, and subjected totechniques such as site-directed mutagenesis to improve further thebinding affinity for the desired protein. Through appropriate geneticengineering techniques, a peptide optimized for binding in this mannercan be introduced into a high-expression host cell (e.g., a bacterialhost such as E. coli), optionally produced as a cleavable fusionprotein, and isolated in high yield. In this manner, large amounts of apeptide second agent can be prepared and linked, through standardtechniques, to a genotoxic first agent to produce the heterobifunctionalcompound disclosed herein. Appropriate techniques for linking peptidesecond agents to genotoxic first agents without incurring substantialloss of protein-recognition capability or genotoxicity are known andavailable.

Still another general class of second agents 9 include organic andinorganic compounds isolated from libraries of synthetic organic andinorganic compounds prepared by combinational synthesis (Needels et al.(1993), 90 P.N.A.S. USA 10700-10704; Ohlmeyer et al. (1993), 90 P.N.A.S.USA 10922-10926).

Linkage Between First and Second Agents

In the heterobifunctional compounds disclosed herein, theabove-described first and second agents are linked together, preferablycovalently. In many embodiments, the first and second agents are linkedthrough covalent linker 7. In other embodiments, linkage of the firstand second agents is accomplished by noncovalent association. In suchembodiments, the first and second agents optionally become linked toform compound 3 intracellularly. Linkage thus can occur after either thefirst agent has bound to cellular DNA, or the second agent has becomecomplexed with the cell component. One example of a noncovalent linkercomprises complimentary oligonucleotide strands (e.g.,oligo(dG)/oligo(dC)) covalently attached, respectively, to the first andsecond agents.

In most embodiments, however, the first and second agents are linkeddirectly by a covalent bond or indirectly through covalent bonds to anorganic linker. This organic linker comprises a linear, branched orcyclic, aliphatic, aromatic or mixed aliphatic and aromatic organiccompound comprising up to about 20 carbon atoms. The organic linker canbe, for example, a peptide, oligosaccharide, oligonucleotide, carbamateor urea compound, such as an oligocarbamate peptide analog. Additionalexamples of linkers include polymers assembled from linkable monomersindependently selected from ethyleneglycols, alkyldiamines and the likesuch as polyethylene glycol, ureas, or spermine/spermidine. The linkerserves to space apart the binding moieties of the first and secondagents such that the heterobifunctional compound disclosed herein cansterically accomodate concurrent binding to cellular DNA and the cellcomponent. Yet, the linker does not separate the first and second agentsso far as to obviate shielding of the genomic lesion by the cellcomponent that is bound to the second agent. Preferably, then, theorganic linker comprises up to about 10 carbon atoms. More preferably,it comprises up to about 5 carbon atoms. Whether covalent linkage of thefirst and second agents is direct or indirect (through the optionallinker), the linkage is stable under physiological conditions,particularly intracellular conditions. That is, the linkage is resistantto cleavage by hydrolysis or other biochemical processes, includingenzymatic processes. For this reason, linkers comprising amide or esterbonds are not presently preferred. Conversely, linkers comprisingcarbamate or urea moieties are preferred herein due to their stabilityand hydrophilicity characteristics. For example, oligocarbamate peptideanalogs comprised of aminocarbamate monomers linked through a carbamatebackbone have been reported to be stable for at least 150 minutes in thepresence of trypsin or pepsin (Cho et al. (1993), 261 Science1303-1305).

Linkage of the first agent to the second agent, either directly orthrough the optional organic linker, can be accomplished by applyingroutine chemical or biochemical techniques, or modifications thereofthat will be readily apparent to those of skill in the art. Theparticular linkage reactions carried out will be determined by the typesof first and second agents to be joined to produce a desiredheterobifunctional compound.

Uses For Heterobifunctional Programmable Genotoxic Compounds

The heterobifunctional programmable genotoxic compounds disclosed hereinare useful in a method for destroying selected cells in a heterogenouscell population. Broadly, the method comprises the steps of contactingthe heterogenous cell population with a heterobifunctional compound asdisclosed above, and incubating the cell population with the compoundfor a period of time sufficient for the compound to internalize withincells, bind to cellular DNA and bind to a cell component preferentiallyassociated with selected cells so as to produce a steric shield thatprotects genomic lesions from repair. As mentioned previously, theheterogenous cell population can comprise cells of a unicellular ormulticellular organism, and can comprise cells maintained in culture,cells withdrawn from a multicellular organism, or cells present in thetissues or organs of a multicellular organism. That is, the method canbe practiced in vitro, ex vivo (using a sample, such as a biopsy,withdrawn from a multicellular organism such as a mammal, e.g., ahuman), or in vivo, by local or systemic administration to amulticellular organism. The recognized cell component can be onenaturally associated with the cell, or one intentionally introduced intothe cell, e.g., by genetic engineering techniques. The present methodtherefore offers the prospect of broadening the range of biologicalselection methods available, e.g., for the production of recombinantproteins or for the isolation of cells with improved or desirablecharacteristics.

Extensive discussion has been devoted herein to programmable genotoxiccompounds that are appropriate for co-opting cell components thatphenotypically distinguish, for example, dividing cells such astransformed (malignant or neoplastic) cells from normal cells, virallyinfected cells from uninfected cells, and cells of a pathogenic organismfrom cells of a host organism. It should be understood that the methoddisclosed herein can be practiced to achieve the selective killing ofcells that are phenotypically distinguishable from other cells of aheterogenous cell population on any of these grounds. In particular itshould be understood that the method can be used to achieve selectivekilling of neoplastic (transformed) cells of colorectal, reproductivetract, hepatic, lymphoid, mammary, myeloid, neurologic or respiratorytract origin. Cells that are of reproductive tract origin can be morespecifically, of ovarian, uterine, endometrial, cervical, vaginal,prostate, or testicular origin. Cells that are of mammary origin can bemore specifically, of breast origin. As is apparent from the disclosureherein, selective killing of such cells can be accomplished through theuse of second agents that recognize intracellular proteins associatedwith malignant transformation. Thus, for example, heterobifunctionalcompounds can be programmed or designed to selectively destroy malignantcells that express an oncogene product (e.g., erbA, able or myc) amutant tumor suppressor gene product (e.g., mutant p53) or an aberrantcyclin or cyclin-dependent kinase. Appropriate heterobifunctionalcompounds would comprise a genotoxic agent linked to an aptamer, bindingpolypeptide or small organic molecule produced by combinatorialsynthesis, that binds the target macromolecule. Alternatively, compoundscan be programmed to selectively destroy cells whose survival orproliferation are dependent on the expression of certain genes byincorporating a second agent that is a transcription factor decoy.Malignant cells whose proliferation is driven by an aberrantly expressedligand-responsive transcription factor, such as an estrogen receptor,androgen receptor or progesterone receptor, can be selectively destroyedby compounds incorporating ligand mimics as second agents. Such ligandmimics include androgens, estrogens, progesterones, glucocorticoids andreceptor binding analogs and derivatives thereof (e.g., the clinicallyrelevant estrogen analog, tamoxifen). For example, estrogen-containingheterobifunctional compounds can be used to achieve selective killing ofbreast carcinoma cells, progesterone compounds can be used similarly tokill uterine or endometrial cancer cells, and androgen compounds can beused to kill prostate cancer cells.

Heterobifunctional compounds of the present invention also can bedesigned to destroy selectively cells of an infectious organism, eitherin vitro or in vivo, that are present in a heterogenous cell populationcomprising cells of a host or infected organism, and cells of aninfectious organism such as a bacterium, a fungus, a virus or aparasite. Thus, compounds disclosed herein are expected to beparticularly useful in maintaining the health and integrity of culturedcells (e.g., mammalian cells) in vitro, as well as in the treatment ofinfectious diseases in vivo caused by pathogenic organisms includingthose with acquired resistance to currently available antibiotic,antifungal or antiparasitic drugs. Infectious diseases for which theavailability of fatal engineered compounds are urgently neededaccordingly include, but are not limited to, septic wound infections,hospital-acquired infections, tuberculosis, malaria and amoebicdysentery. Other examples, particularly of parasitic diseases, for whichprogrammable genotoxics offers the potential to expand the range ofavailable genotoxic agents, include schistosomiasis, filiariasis, Chagasdisease, leishmaniasis, sleeping sickness, toxoplasmosis,pneumocystosis, giardiasis, trichomoniasis, croptosporidiosis, and thelike. Harrison's Principles of Internal Medicine, Part 5 InfectiousDiseases, Ch. 156-172. Certain of these diseases are relatively commonamong cosmopolitan communities, while others present severe threats tothe populace of developing nations.

Alternatively, the present compounds can be used in vitro to enrich aheterogenous cell population for cells having a desirablecharacteristic, or cells lacking an undesired characteristic. Thus, thepresent compounds offer new alternatives to current methods for, e.g.,isolating a hybridoma cell producing a desired antibody from aheterogenous cell population comprising primary antibody producing cellsand an immortalized fusion partner cell line. Alternatively, the presentcompounds expand the range of genetic selection agents useful forseparating a desired cell transfected with heterologous nucleic acidsfrom a cell population comprising unsuccessful transfectants.

Those of skill in the art will readily understand and appreciate thatthe incubation period needed to achieve selective cell killing will varywidely, depending on the circumstances under which the invention ispracticed. In many instances, the time period needed to achieveselective killing of cultured cells or suspensions of unicellularorganisms or of cells withdrawn from a multicellular organism will beless than the time needed to achieve selective killing of cells in vivoin a multicellular organism. For in vivo use to destroy selected cellsin the tissues of a multicellular organism (e.g., a mammal) theprotocols in which the drugs are used will vary depending on thelocation of cells to be destroyed, replicative rate of the cells, levelof repair proficiency of the cells, dose of heterobifunctional compoundadministered, route of delivery of the compound (generally eithersystemic or local, and either enteral or parenteral), andpharmacokinetic profiles of clearance and tissue uptake of the compound.Variables affecting the dose needed thus include, but are not limitedto, the nature (e.g., species or tissue type), quantity andaccessibility (i.e., body compartment location) of selected cells to bedestroyed, and the nature, genotoxicity, and affinity of the compoundfor recognized cell component. The present compound can be combined witha pharmaceutically acceptable carrier or excipient for formulation as aliquid, suspension, solid, salve, ointment or the like, suitable fororal, nasal, intravenous, intraperitoneal, topical, subdermal,intramuscular, or other routes of administration. The present compoundcan be administered in a single dose (e.g., a bolus injection), a seriesof doses of equivalent, escalating, decreasing or intermittently variedquantity, or infused over a period of time (e.g., by intravenous drip orinfusion), or by release from a slow-release delivery vehicle. Theappropriate dose of the present compound will of course be dictated bythe precise circumstances under which the invention is practiced, butwill generally be in the range of 0.01 ng to 10 g per kg body weight,preferably in the range of 1 ng to 0.1 g per kg, and more preferably inthe range of 100 ng to 10 mg per kg.

If desired, the degree of selective cell killing achieved can beascertained through standard, widely available techniques, such asvisual or microscopic inspection, biochemical, chromogenic orimmunologic methods for detecting products of selected cell lysis, andthe like. Such techniques can be used to establish both the dose andtime period effective to accomplish objectives of the present inventionunder particular circumstances. Once effective doses and time periodsare established, it may be no longer necessary to monitor the progressof selective cell killing.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

EXAMPLE 1 Western and Southwestern Blotting Studies with hUBF

Probe Preparation. The DNA probe used for southwestern blotting was a422 base pair (bp) Aval restriction fragment excised from M13mp19replicative form DNA. Platinated probes were prepared by treating theAval-digested DNA with cisplatin or trans-DDP, and the formal bounddrug/nucleotide ratios (r_(b)) were determined by using atomicabsorption spectroscopy as described in Donahue et al. (1990), 29Biochemistry 5872-5880.

Western and Southwestern Blotting Technique. HeLa whole cell extracts(WCE) were prepared by the sonication procedure of Samson et al. (1986),83 Proc. Natl. Acad. Sci. U.S.A. 5607-5610. The 97 kDa hUBF species wassynthesized by in vitro transcription and translation from the plasmidpTβGUBF1 as reported in Jantzen et al. (1992), 6 Genes & Dev. 1950-1963.In vitro translated hUBF was quantitated by the incorporation of ³⁵S-methionine. Protein samples (75 μg WCE or 8 ng hUBF) were resolved on5-15% gradient SDS polyacrylamide gels and transferred to nitrocellulosemembranes. Parallel blots of HeLa whole cell extracts (WCE) and in vitrotranslated hUBF (hUBF) were probed with various ³² P labeled DNAfragments (southwestern analysis, panels A-C of FIG. 2) or antiserumagainst hUBF (Anti-NOR-90) (panel D of FIG. 2). For southwesternanalysis, the air dried membranes were processed as reported in Toney etal. (1989), 86 Proc. Natl. Acad. Sci. U.S.A. 8328-8332. In the probingstep, the labeled DNA was present at about 5×10⁴ cpm/ml and thenonspecific competitor poly(dI-dC) * poly(dI-dC) at 5 μg/ml. The blotshown in Panel A was probed with the cisplatin (cis-Pt-422) modifiedprobe, that shown in Panel C with thetrans-diamminedichloroplatinum(II)(trans-Pt-422) modified probe, andthat shown in Panel B with unmodified (Un-422) probe. HeLa proteinsrecognizing cis-Pt-422 are listed by molecular weight to the left ofPanel A. The r_(b) values for probes modified by cisplatin and trans-DDPwere 0.043 and 0.052, respectively. During autoradiography, a 0.254 mmthick copper sheet was used to block ³⁵ S emissions selectively from thein vitro translated hUBF. For western analysis (Panel D), the filter wasprobed with a 1/250 dilution of antiserum to human NOR-90 (hUBF)obtained as a gift from E. K. L. Chan (Chan et al. (1991), 174 J. Exp.Med. 1239-1244). Antibody binding was visualized through standardtechniques, using a chemiluminescent detection system commerciallyavailable from BioRad. The positions of both HeLa and in vitrotranslated hUBF are shown. A 120 kDa species of unknown identity wasalso visualized in the WCE with Anti-NOR-90.

Results. Protein blots of human HeLa cell extracts (FIG. 2A) probed withcisplatin modified DNA (southwestern analysis) revealed species ofM_(r)(app) 97, 94 and 28 kDa. Unmodified DNA or DNA modified with theclinically ineffective trans-DDP compound was not bound by theseproteins (Panels B and C), although a 105 kDa nonspecific DNA bindingprotein was detected with each of the three DNA probes. The 28 kDaspecies has recently been identified as the abundant chromatin proteinHMG1 (Pil and Lippard (1992), 256 Science 234-237; Hughes et al. (1992),J. Biol. Chem. 13520-13527). The precise functions of HMG1 remainunclear although it has been proposed to play roles in the maintenanceof chromosome structure and the alteration of DNA topology, and maytherefore be important for transcription and DNA replication (Bustin etal. (1990), 1049 Biochim. Biophys. Acta 231-243). Since the HHG box is aunifying feature of many cisplatin lesion recognition proteins, it waspostulated that the 97 and 94 kDa proteins possess this DNA bindingdomain. The RNA polymerase I transcription factor hUBF contains severalregions of homology to HHG1 (Jantzen et al. (1990), 344 Nature 830-836)and exists as both 97 and 94 KDa species owing to an alternativesplicing event (Chan et al. (1991), 174 J. Exp. Med. 1239-1244). Westernblot analysis with hUBF antiserum demonstrated that the hUBF doubletresembles the bands detected by southwestern analysis (compare FIG. 2,Panels A and D). From these observations, it was postulated that hUBFbinds to cisplatin DNA lesions. This postulate was confirmed bysouthwestern blot analysis of in vitro translated hUBF (FIG. 2A, lane2).

EXAMPLE 2 DNase I Footprinting Studies of the hUBF-Cisplatin! Complex

Probe Preparation. A 100 bp DNA fragment containing a single, centrallylocated 1,2 intrastrand cis- Pt(NH₃)₂ !²⁺ d(GpG) crosslink (G G-100) andthe analogous unmodified fragment (Un-100) were used as both competitorDNAs and probes in hUBF footprinting experiments. These DNA fragmentswere kindly provided by P. Pil and S. J. Lippard (Pil and Lippard(1992), 256 Science 234-237). The adduct-containing strand of G G-100and the analogous unmodified strand of Un-100 were 5' end-labeled withγ³² P-ATP (>6,000 Ci/mmole), using polynucleotide kinase according tostandard procedures. The 5' end of the unadducted strand was removedwith Aval to generate the 90 bp footprinting probes. These were purifiedby passage through Sephadex G-25 Quickspin™ columns (BoehringerMannheim).

DNase I Footprinting Technique. Homogeneous HeLa hUBF was used togenerate DNase I footprints on both rRNA promoter (described below) andplatinated DNA probes. Footprinting was performed essentially asdescribed in Bell et al. (1988), 241 Science 1192-1197. hUBF was addedto footprinting reactions containing the appropriate labeled DNA probe10³ -10⁴ cpm, 0.7-50 pH, depending on the experiment) and binding buffer(25 mM Tris-HCl pH (7.9), 14 mM MgCl₂, 0.5 mM dithiothreitol, 10%glycerol, 50 mM KCl, 0.05% Nonidet-P40, 2.5 mM CaCl₂) in a total volumeof 50 μl. The binding reactions were incubated for 10 min. at 30° C. andthen digested with DNase I (Worthington DPFF grade) for 1 min. at 25° C.The DNase I reactions were terminated by adding a solution of 20 mMEDTA, 1% SDS, 0.2M NaCl, and 50 μg/ml yeast total RNA. Samples werephenol/chloroform extracted, ethanol precipitated, and electrophoresedaccording to standard procedures on denaturing wedged (0.4-1.5 mm)sequencing gels (6% or 12% for promoter footprints or G G-100footprints, respectively) at 70W. Gels were fixed, dried and exposedwith an intensifying screen to preflashed X-ray film at 80° C., andanalyzed by using a Molecular Dynamics PhosphorImager™ imaging machine.

Results. As shown in FIG. 3, Panel A, 400 pm hUBF was sufficient toprotect the area of the probe immediately adjacent to the defined G Gadduct from DNase I cleavage (compare lanes 1 & 2). A distinctprotection pattern was observed in the 14 bp region encompassing theadduct, providing direct evidence that hUBF recognizes the structuraldistortion induced by G G. The relevant sequence is shown to the left,and the protected residues are displayed within the box. The broken lineindicates a residue immediately 5' to G G that remained DNaseI-sensitive. The established structural features of the G G adductinclude helix bending (34°) toward the major groove (Bellon and Lippard(1990), 35 Biophys. Chem. 179-188) and unwinding (-13°) (Bellon et al.(1991), 30 Biochemistry 8026-8035). No such protection is afforded theanalogous unmodified 100-mer (lane 3), which gave the same cleavagepattern both in the presence and in the absence of hUBF (lanes 3 & 4).Cleavage patterns of G G-100 and Un-100 near the cisplatin adduct shouldbe directly comparable (lanes 2 & 4). Panel B shows the PhorphorImagersemiquantitative profile of hUBF binding to G G-100. Y is the fractionalsaturation of G G-100 and was estimated by monitoring the intensity ofthree bands in the protected region at each hUBF concentration. The datafit the equation K_(d) = hUBF! G G-100!/ hUBF-G G-100! when K_(d) =60pM. The protein concentration giving half-maximal binding (K_(d)(app) isindicated by the broken line. The labeled probe was present at 20 pM(10⁴ cpm). These results indicate that hUBF-cisplatin! complex formationis exceptionally favorable, in energetic terms. From the shape of thesemiquantitative binding profile, it is also apparent that binding isnon-cooperative.

EXAMPLE 3 DNase I Footprint Studies of the hUBF-DNA Promoter! Complex

Probe Preparation. For footprinting studies, the EcoRI-BstE11restriction fragment of pSBr208 containing the -208 to +78 region of thehuman rRNA gene was either 5' or 3' end-labeled on the noncoding strand.pSBr208 was digested with EcoRI and the 5' phosphate was removed withcalf intestinal phosphatase. EcoRI-digested pSBr208 was 5' end-labeledwith γ³² P-ATP (>6,000 Ci/mmole) and subsequently digested with BstE11.The 286 bp footprinting probes were purified on 5% polyacrylamide gelsand electroeluted. In cases where higher specific activity footprintingprobes were required, the noncoding strand was 3' end-labeled by usingthe Klenow enzyme in the presence of α-³² P!-dATP, α-³² P!-dCTP, andα-³² P!-dGTP (>6,000 Ci/mmole).

The DNase I footprinting technique described in the preceding examplewas followed in the present promoter-binding studies.

Results. The biological significance of cisplatin adduct recognition byhUBF ultimately depends on the affinity of the interaction. Theinteraction of hUBF with rDNA accordingly provides a useful benchmarkvalue for a biologically relevant affinity. The upper panel of FIG. 4shows the rDNA binding profile at hUBF concentrations ranging from 7-78pM. The formation of hUBF-promoter! complexes resulted in DNase Ihypersensitivity at positions -20 and -95 in the CORE and UCE elements,respectively. In addition, the 40 bp region that symmetrically flanks-95 became refractory to cleavage (Bell et al. (1988), 241 Science1192-1197). The degree of promoter occupancy was most easily visualizedby the increased DNase I sensitivity of the -95 position in the upstreamcontrol element (UCE). The 3' labeled probe used to generate the resultsshown in FIG. 4 was present at 0.7 pM (10³ cpm). Bands thus appear asdoublets due to incomplete labeling.

hUBF binding was next quantitated by measuring the intensity of theenhanced cleavage at -95. In the bottom panel, intensity is reported tothe left in arbitrary PhosphorImager units (PIU), and, to the right, isexpressed as the apparent fractional saturation (Y). The proteinconcentration giving half-maximal binding (K_(d)(app), 18 pM) isindicated by the broken line. Thus, hUBF binds tightly to its endogenoussite(s) in the rDNA promoter. It should be noted that the affinities ofhUBF for promoter sequences and for the cisplatin decoy are comparable,differing by only three-fold. This suggests that cisplatin adducts canbe effective decoys for hUBF in the cellular milieu. It should furtherbe noted that the UCE footprint qualitatively resembles that observedfor G G-100. In both complexes, a protected region symmetrically flanksa nuclease sensitive site. The shape of the hUBF-promoter bindingprofile reveals that the fraction of bound promoter (Y) increasessharply over a narrow range of hUBF concentrations, indicating thatbinding is cooperative. A Hill plot of these data yielded a best fitline (r=0.997) with a Hill constant (n_(H)) of 2.7, indicating positivecooperativity. Cooperativity has also been reported for Xenopus UBFbinding to enhancer repeats (Putnam and Pikaard (1992), 12 Mol. CellBiol. 4970-4980). An important consequence of cooperativity in thecontext of the transcription factor hijacking model is that a smalldecrease in the pool of free nuclear hUBF can strongly decrease promoteroccupancy.

EXAMPLE 4 Competitive Inhibition of hUBF-rDNA Promoter! Complexing byCisplatin Decoys

From the comparable values of the hUBF affinity constants for cisplatinadducts and the rDNA promoter, it seemed likely that cisplatin adductsshould be effective competitive inhibitors of hUBF-promoter! complexformation. Accordingly, a competition study was carried out, using theprobe preparation and DNase I footprinting techniques discussed in thepreceeding examples.

Competitive Technique. Purified HeLa UBF was added to all samples,except the negative control (shown in lane 1 of FIG. 5), to a finalconcentration of 160 pM. This level of hUBF is safely above thatproducing an apparent fractional saturation (Y) of 1 in the positivecontrol (lane 2). The 5' labeled probe was present at 46 pM (10⁴ cpm).Un-100 (lanes 3-6) and G G-100 (lanes 7-12) were added as unlabeledcompetitors to the final concentrations (nM) listed. The competitiveeffect was estimated by measuring Y of the promoter probe. Y values areshown at the bottom. Lanes 1 and 2 were used as standards to calculate Yin lanes 3-12.

Results. FIG. 5 shows that G G-100 efficiently antagonized hUBF-promoterinteractions. The reduced intensity of bands at positions -21 and -95 inthe CORE and UCE elements, and the reappearance of bands betweenpositions -75 and -115 illustrate this effect (lanes 7-12). At asaturating concentration of hUBF, the formation of promoter complexeswas completely inhibited by a platinum adduct concentration of 5×10⁻⁹ M(lane 11), which is well below the adduct levels in cancer patient DNA(10⁴ -10⁵ /cell, or 10⁻⁷ -10⁻⁶ M)(Reed et al. (1993), 53 Cancer Res.3694-3699). The corresponding unmodified competitor DNA (Un-100) was a10-30 fold weaker competitor of hUBF than G G-100 (lanes 3-6). SinceUn-100 contains up to 100 overlapping nonspecific binding sites comparedto the one specific binding site in G G-100, the preference of hUBF fora platinated versus an unplatinated site may be as high as 1-3×10³ fold.These results directly support the view that cisplatin decoyseffectively hijack hUBF, sequestering this transcription factor awayfrom its endogenous genomic binding site and leaving the rDNA promoterunoccupied. From these results, disarray of the cellular proteinsynthesis machinery can be predicted.

EXAMPLE 5 Demonstration that a Heterobifunctional Compound can MediateBinding of a Chosen Protein to a Genomic Lesion Site

TMP-biotin Conjugate. A 17-mer oligonucleotide, referred to a U-17, wassynthesized by standard phosphoramidite chemistry. U-17 comprised asingle, centrally-located 5'-TA-3' site, along with a uracildeoxynucleotide located three bases away from the TA site on the 3'side. The oligomer was purified on a 20% denaturing (7M urea)polyacrylamide gel (acrylamide/bis, 19:1) and electroeluted by using anAmicon centrilutor. Urea was removed from the oligomer by severaldistilled water washes in Amicon Centricon 3™ microconcentrators.Purified U-17 was 5'-end labeled with γ⁻³² P! ATP (6000 Ci/mmole, NewEngland Nuclear) by using T4 polynucleotide kinase (New England Biolabs)according to standard techniques. Unincorporated label was removed bycentrifugation through a pre-packed G-25 column (Boehringer-Mannheim).Labeled U-17 was then annealed to its unlabeled complementary strand.TMP-biotin conjugate (dissolved in 50% (v/v) acetonitrile) was thenadded to the duplex oligmer solution with the molar ratio of TMP-biotinto base pair at about 1000:1. After being incubated at room temperaturefor 10 min, the mixture was placed on a chilled surface and subjected tonear UV irradiation with a 15-W General Electric lamp (maximum output at365 nm). The final irradiation dose was about 85 kJ/m². The resultingirradiated mixture, now comprising TMP-biotin lesioned U-17, wasseparated on a 20% denaturing polyacrylamide gel. A gel slice containingmonoadducted TMP-biotin U-17 strand was cut out, and the lesioned DNAwas purified by electroelution as described above. Finally, the lesionedU-17 strand was annealed to its cognate unlabeled complementary strandto form the double-stranded lesioned probe.

Gel Mobility Shift Assay. The binding of streptavidin to U-17monoadducted with the TMP-biotin conjugate was measured by incubatingthe probe with streptavidin (Pierce) in 10 ul of binding buffer 25 mMTris-HCl (pH 7.4), 100 mM NaCl and 1.5 MgCl₂ ! at room temperature for10 min, and electrophoresing the mixture on a 5% nondenaturingpolyacrylamide gel (acrylamide/bis, 29:1) at 4° C. A constant amount ofthe lesioned U-17 (3200 cpm, .sup.˜ 0.1 nM) was used in each incubation,with the concentration of streptavidin varied from 0 to 50 nM. Freed-biotin (0.4 mM) was added into the incubation(s) where indicated.After electrophoresis, the gel was dried and exposed to x-ray film withan intensifying screen. The dried gel was also exposed to aPhorphorImager screen and the data were analyzed with IMAGEQUANTsoftware (Molecular Dynamics, Sunnyvale, Calif.).

Results. Streptavidin retarded the electrophoretic mobility of U-17fragments monoadducted with the TMP-biotin conjugate (FIG. 6, Panel A).The retardation was caused by the binding of streptavidin to the biotininserted into the DNA because free biotin reversed the retardation,presumably by competing with the immobilized biotin for streptavidin(lane 8 in Panel A). Quantitation of the data by IMAGEQUANT software ofMolecular Dynamics gave rise to a binding curve (Panel B). Thestreptavidin concentration for the half-maximum binding (C_(1/2)) wasabout 1.5 nM, suggesting that the K_(d) between streptavidin and theimmobilized biotin was also about 1.5 nM. Streptavidin showed littlebinding activity to either U-17 oligomer or U-17 monoadducted with justa psoralen derivative (data not shown). It should be pointed out thatthe K_(d) value differs quite significantly from that of free biotinwith streptavidin or avidin. As discussed previously, the observedincrease of this K_(d) value was possibly attributable to steric effectsexerted on the streptavidin-biotin binding by the adduction ofTMP-biotin conjugates to DNA.

EXAMPLE 6 Demonstration that Lesion-Bound Streptavidin Hinders Access bya DNA Repair Enzyme

Uracil Glycosylase Protection Assay. Double-stranded, TMP-biotinmodified U-17 obtained as described earlier was used as the probe inthis assay. The probe (4000 cpm, .sup.˜ 0.15 nM for each reaction) wasincubated first in 12 ul of glycosylase buffer 30 mM Tris.HCl(pH 7.4),50 mM KCl and 5 mM MgCl₂ ! at room temperature for 10 min in thepresence of streptavidin (36 ng, .sup.˜ 50 nM) where indicated. In someincubations, 0.4 mM free d-biotin was added. After the incubation, 3 ul(0.15 units) of uracil glycosylase (Boehringer-Mannheim) was added, andthe mixtures were then incubated at 37° C. for 5 min to 40 min. At theend of each incubation, 85 ul freshly prepared 1.25M piperidine (Fisher)was added, and the samples were subsequently heated at 90° C. for 1 hr.Since an apurinic site in a DNA molecule is labile to alkali cleavage,Lindahl and Andersson (1972), 11 Biochemistry 3618-3623, piperidinetreatment as stated above would have resulted in DNA strand breaks ifany apurinic sites were generated from the uracil glycosylase treatment.The samples were vacuum centrifuged to remove the piperidine and washedby resuspension in distilled water and followed by vacuum centrifugationagain. Washed samples were finally resuspended in denaturing loadingbuffer 80% (v/v) recrystallized formamide, 0.1% (w/v) xylene cyanol and0.1% (w/v) bromphenol blue!, analyzed on a 20% denaturing polyacrylamidegel. The gel was exposed (without being dried) to an x-ray film with anintensifying screen at -80° C.

Results. As indicated in FIG. 7, streptavidin, when complexed with theTMP-biotin DNA adducts, inhibited the removal of a nearby uracil base bythe uracil glycosylase (compare lanes 3-6 with lanes 7-10). Theinhibition was substantially reversed when free biotin was added (lanes11-14). The TMP-biotin DNA adducts were stable even after being heatedat 90° C. for 1 hr (lane 1). A small fraction of the TMP-biotin adductswas removed when the probe was subjected to piperidine treatment band(b) in lane 2; bands (b) and bands (d) in lanes 3-14!.

EXAMPLE 7 Demonstration that Lesion-Bound Streptavidin Acts as a StericShield

DNase I Protection Assay (Also Called DNase I Footprinting). Again, ³² Pend-labeled double-stranded U-17 modified with the TMP-biotin was usedin this assay. Briefly, the lesioned DNA (5000 cpm, .sup.˜ 0.15 nM) wasfirst incubated with various amounts of streptavidin (0-50 nM) at roomtemperature for 10 min in 10 ul of binding buffer 25 mM Tris-HCl (pH7.4), 100 mM NaCl and 1.5 mM MgCl₂ !. Where indicated, 0.4 mM freed-biotin was included in one of the incubations. At the end of eachincubation, 2 ul of 2.5 mg/ml freshly diluted DNase I (WorthingtonEnzymes, Freehold, N.J.; final concentration, 0.4 mg/ml) was added, andthe digestion was carried out at room temperature for 2 min before beingquenched by the addition of 50 ul of stop solution 20 mM EDTA (pH 8.0),1% SDS and 50 ug/ml yeast total RNA!. The samples were then precipitatedby ethanol. After being washed once with 80% ethanol, the DNA pelletswere air dried and then resuspended in denaturing loading buffer.Finally, the resuspensions were loaded onto a 20% denaturingpolyacrylamide gel. The gel was dried, and exposed to an x-ray film withan intensifying screen at -80° C.

Results. As shown in FIG. 8, when streptavidin was added, the modifiedU-17 became resistant to DNase I cleavage. Enhanced C¹⁶ and G¹⁷ bandsindicated that streptavidin protected these fragments (full-length orone base less) from being further cleaved by DNase I. Since the lesionedDNA probe was 5' end-labeled, and fragments shorter than 5-mer could notbe recovered by the ethanol precipitation used in the experimentalprocedures, only two bases (C⁵ and G⁶) on the 5' side of the modifiedthymidine were observed to be covered by streptavidin. On the 3' side,however, the covered region was more extensive. As discussed above, theprotected region extended at least to the full-length of the probe,which was ten nucleotides away from the modified base. It is reasonableto expect that a similar length on the 3' side of the probe is alsoprotected by streptavidin. These observations suggested thatstreptavidin, a protein of about 50 kD, covered at least twentynucleotides flanking the thymidine where a TMP-biotin was monoadducted.DNA excision repair enzymes in mammalian cells, which are able to repaira variety of DNA damages (Friedberg (1985), DNA Repair) repair a DNAlesion first by recognizing it and then excising a "patch" or DNAfragment of 27- to 29-nucleotides flanking the lesion site on thedamaged strand (Huang et al. (1992), 89 P.N.A.S. USA 3664-3668; Svovodaet al. (1993), 268 J. Biol. Chem. 1931-1936). It was noted that, even inthe absence of streptavidin, a small region of 4-5 nucleotides on the 3'side of the modified thymidine resisted DNase I cleavage. It is possiblethat this effect was due to the presence of the TMP-biotin lesionitself. A similar effect would not be observed had the experiment beenconducted with an appropriate repair enzyme instead of DNase.

Prospective Example 8: Guidelines For Demonstrating That aHeterobifunctional Compound Forms Genomic Lesions

The ability of a given programmed genotoxic, such as thenitrogen-mustard-steroid decoy, estrogen-chlorambucil, to damage DNA canbe assessed by determining the ability of the compound to forminterstrand crosslinks between opposing strands of a duplex DNAmolecule. Formation of such crosslinks is known to correlate stronglywith the clinical efficacy of bifunctional mustards includingchloramubicil and melphalen (Ross et al. (1978), 38 Cancer Res.1502-1506; Zwelling et al. (1981), 41 Cancer Res. 640-649). The assay isboth simple and rapid. It is based on the separation of DNA strandsunder denaturing conditions of heat and chaotropic compounds (e.g.,urea), or organic solvents (e.g., N,N-dimethylformamide). Whencrosslinked, denatured DNA strands are unable to separate andconsequently migrate more slowly than uncrosslinked separated strandsduring electrophoresis in polyacrylamide gels. After incubating thechosen compound with a short DNA duplex molecule labeled with ³² P, thepercentage of crosslinked DNA molecules can be determined followingseparation by gel electrophoresis according to standard methods.Conditions for crosslinking and gel analysis of both short DNA fragments(Rink et al. (1993), 115 J. Amer. Chem. Soc. 2551-2557) and longer DNAfragments (Hartley et al. (1991), 193 Anal. Biochem. 131-134; Holley etal. (1992), 52 Cancer Res. 4190-4195) have been described in detail.These methods can be adapted to assess the crosslinking of DNA fragmentsranging in size from, e.g., duplex 17-mer oligonucleotides such as U-17,to the 166, 235, 540, 1423, and 3199 base pair fragments obtained fromDde I restriction endonuclease digest of the widely available pGEMplasmid.

Crosslinking capacity of a particular programmed heterobifunctionalcompound should be compared to that of the parent genotoxic agent (e.g.,chlorambucil). The compound under investigation preferably has abilityto produce interstrand crosslinks in DNA in vitro that are comparable tothose of the parent compound under similar conditions. If this is notthe case, the reactivity of synthetic intermediates can be examined todetermine what modification(s) to the parent genotoxic agent's structureis responsible for its reduced crosslinking activity. With thisknowledge in hand, the structure of an optional linker or othercomponent can be modified, if necessary, to restore reactivity of thegenotoxic agent portion of the heterobifunctional compound to a desiredlevel.

Prospective Example 9: Guidelines For Demonstrating That aHeterobifunctional Compound Binds to a Chosen Cell Component

Tight association between the programmed genotoxic,estrogen-chlorambucil, steroid ligand and the estrogen receptor proteinis relevant to the compound's intended function. The strength ofassociation of the ligand decoy receptor complex should be measured forboth the "free" form of the compound, and the form that is covalentlybound to DNA forming a genomic lesion. Knowledge of the interaction ofthe free form of the compound with the receptor can indicate whether theposition of chemical attachment of the steroid ligand to the optionallinker has preserved capacity to interact with the receptor protein.Comparison of the strengths of association of the free and DNA-boundforms of the compound should indicate whether or not the DNA moleculesterically impedes the formation of lesion-shielding complexes.

One of several routine and widely used assays can be employed formeasuring the ability of the free compound to displace a natural steroidligand from the estrogen receptor. Typically, radiolabeled estradiol isfirst bound to the receptor protein in a cell extract prepared fromestrogen responsive tissue such as uterus. Calf uterus is most commonlyused for this purpose. Increasing concentrations of the compound underinvestigation are then added. The amount of estradiol remaining tightlyassociated with the protein as a function of the increasingconcentration of the other chemical provides a measure of the relativeaffinities of the natural and synthetic ligands for the receptor.

Where the compound under investigation has first been covalentlyattached to DNA, its association with the receptor protein can beinvestigated by gel electrophoresis using a routine adaption of mobilityshift techniques described fully in Carthew et al. (1985), 43 Cell439-448. DNA-receptor complexes can thus be electrophoretically resolvedfrom lesioned, uncomplexed DNA through application or routine adaptionof this technique. Furthermore, the strength of the association can bemeasured by addition of competing ligands for the receptor as describedin the previous examples. Increasing amounts of estradiol, for example,would compete with the DNA bound ligand for the receptor protein andthereby restrict formation of the DNA-receptor complex. Theeffectiveness of estradiol in preventing the formation of theDNA-receptor complex should provide a useful measure of their relativestrengths of association with the receptor.

From the results of biomolecular studies such as those described above,it should be possible to predict the effectiveness of compounds underinvestigation for blocking repair of lesions in living cells.

Prospective Example 10: Guidelines for Demonstrating Efficacy of LigandDecoy Compounds

Specificity of heterobifunctional compounds, such as theestrogen-chlorambucil decoy, for killing tumor cells that express theestrogen receptor can be tested readily in available cell culture modelsfor breast cancer. Results derived from these models will formappropriate grounds for reasonably predicting genotoxic effectiveness ofcandidate programmed heterobifunctional compounds for use in vivo. Thatis, effectiveness of the compounds in the present cell culture modelswill provide an early indication of genotoxic potential in multicellularorganisms, such as mammals, including humans. For present purposes,breast cancer cell lines should be chosen for screening protocolsbecause this form of cancer is the principle target for genotoxic usesof estrogen receptor decoys. Several human breast cancer cell lines arewidely available and have been characterized as to their estrogenreceptor status. The MCF-7 and MDA-MB-231 cell lines are two suchexamples. Estrogen receptor status plays a key role in determining theresponses of these cell lines to estrogens and genotoxic antiestrogenssuch as tamoxifen. Estrogens stimulate the growth of the estrogenreceptor positive cell line MCF-7, while having no effect on the growthrate of MDG-MB-231, which lacks the receptor. Likewise, antiestrogenssuch as tamoxifen inhibit the growth of MCF-7 cells, but have no effecton the growth of MDA-MB-231 cells. These two cell lines therefore allowa determination of whether compounds such as the estrogen-chlorambucildecoy are more effective than chlorambucil itself in killing cells thatcontain the target receptor. Thus, cell lines with high levels ofestrogen receptor protein should be much more sensitive to theheterobifunctional decoy.

Cell sensitivity can be assessed using a growth inhibition assay. Equalconcentrations of chlorambucil and the chlorambucil-estrogen conjugatecan be added to cell cultures, and the rate of cell proliferationdetermined by counting the number of cells in replica cultures up toseven days post treatment. The increase in cell numbers in both treatedand untreated control cultures can be compared to assess potentialantitumor effects. Favorable results should be confirmed by repeatingthe test using a phenotypically different pair of receptor-bearing andreceptor independent breast carcinoma cell lines. Drugs that demonstratea 2-4 fold or greater ability to inhibit the growth of estrogen receptorpositive cells, as compared to receptor negative cells, should beselected for testing in mammals.

EXAMPLE 11 In Vitro Genetic Selection of a Pool of Aptamers that BindSelectively to Mutant P53

Two 10-mer peptides (EP240-Cys: NH₂-Thr-Phe-Arg-His-Ser-Val-Val-Val-Pro-Cys-COOH SEQ ID No: 1; andEP240S-Cys: NH₂ -Thr-Phe-Val-His-Val-Ser-Arg-Val-Pro-Cys-COOH SEQ ID No:2) were synthesized by standard techniques and coupled to aThiol-Sepharose supporting matrix through the cysteine residues in thepeptides. Peptide EP240-Cys comprises the five residue epitoperecognized by PAb 240 shown underlined as thus was used as the selectiontarget peptide. Peptide EP240S-Cys, in which the epitope sequence isscrambled, was used to eliminate aptamers that boundnon-sequence-specifically to the target peptide. The C-terminal cysteineresidues, which do not exist in the native protein sequence, wereattached to both peptides to facilitate immobilization ontothiol-derivitized agarose beads, and elution of the peptides along withthe bound aptamer candidates under reducing conditions (e.g., 20, nMDTT). A pool of 100-Her oligonucleotides containing a central 64-mertotally randomized sequence flanked by 18-mer PCR primer regions at eachend was synthesized by standard techniques. About 90 pmoles of theoligonucleotides, representing no fewer than 10¹³ different molecules,were amplified by about 100-fold by PCR, using a 5' end-biotinylatedprimer for one of the two flanking regions. The unbiotinylated DNAstrand was thereafter isolated by binding the double-stranded PCRproducts to a streptavidin column and eluting the column with 0.15NNaOH. The amplified pool of single stranded candidate aptamers (about900 pmoles) was first applied to a pre-selection column containing thescrambled EP240S-Cys peptides. This step was designed to eliminatenonspecific binding. The DNA that went through the pre-column wasdirectly loaded onto the selection column containing EP240-Cys epitopepeptides. After extensive washing with binding buffer, the selectioncolumn was eluted with binding buffer containing 20 mM DTT. The elutedDNA was subjected to PCR amplification. Rounds of selection andamplification were repeated to generate a pool rich in candidateaptamers having the desired binding property. To date, nine rounds ofselections have been completed. Preliminary results indicate that apopulation of aptamers has been selected that bind preferentially to theselection column EP240-Cys and not to the pre-selection columnEP-240S-Cys. Individual aptamers isolated from this pool will besubjected to assessment of their binding characteristics for mutant p53,and will be further developed as heterobifunctional compounds programmedto selectively destroy cells that express a recognized p53 mutant.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ThrPheArgHisSerValValValProCys    1510    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ThrPheValHisValSerArgValProCys    1510    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..26    (D) OTHER INFORMATION: /product= "NUCLEOTIDE SEQUENCE    SHOWN IN FIGURE 3"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CAGTCTCCTTCTGGTCTCTTCTCAGT26    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /product= "NUCLEOTIDE SEQUENCE    SHOWN IN FIGURE 8"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CGGCCGTACGUGCGCCG17    __________________________________________________________________________

What is claimed is:
 1. A cell membrane permeant heterobifunctionalcompound effective in destroying selected cells in a heterogenous cellpopulation, comprisingi) a nitrogen mustard linked, via a linkage stableunder intracellular conditions, to ii) an agent that mediates selectivebinding of said compound to an intracellular diffusable nuclear proteinthat is preferentially present in said selected cells and is encoded byan oncogene or a mutant tumor suppressor gene, such that athree-membered complex forms between said compound, said protein, andcellular DNA of said selected cells, said complex being effective topreferentially inhibit repair of genomic lesions formed in said selectedcells by binding of said compound to said cellular DNA.
 2. A compound ofclaim 1, wherein said linkage stable under intracellular conditionscomprises a covalent bond.
 3. A compound of claim 1, wherein saidlinkage stable under intracellular conditions comprises an organiclinker comprising up to about 20 carbon atoms.
 4. A compound of claim 3wherein said organic linker comprises up to about 10 carbon atoms.
 5. Acompound of claim 4 wherein said organic linker comprises up to about 5carbon atoms.
 6. A compound of claim 1 wherein said agent binds anuclear protein of endogenous cellular origin.
 7. A compound of claim 1wherein said selected cells are dividing cells.
 8. A compound of claim 1wherein said selected cells are neoplastic cells.
 9. A compound of claim1 wherein said agent binds a nuclear protein encoded by a mutant p53gene.
 10. A compound of claim 9 wherein said agent binds to a mutant p53epitope, NH₂ -Arg-His-Ser-Val-Val-COOH.
 11. A compound of claim 1wherein said agent binds a nuclear protein encoded by an oncogeneselected from the group consisting of: erbA, abl, jun, fos, myc, N-myc,myb, ski and rel.
 12. A compound of claim 1 wherein said agent binds anuclear protein encoded by an oncogene in the genome of a tumorigenicvirus.
 13. A compound of claim 12 wherein said virus is selected from anadenovirus, a papovavirus, a retrovirus, a polyoma virus, a human T-cellleukemia virus, an Epstein-Barr virus, and a papilloma virus.
 14. Acompound of claim 1 wherein said agent is selected from a peptide ligandand a nucleic acid aptamer.